2006 Publications

An Arabidopsis (Arabidopsis thaliana) multigene family (predicted to be more than 20 members) encodes plant C-terminal domain (CTD) phosphatases that dephosphorylate Ser residues in tandem heptad repeat sequences of the RNA polymerase II C terminus. CTD phosphatase-like (CPL) isoforms 1 and 3 are regulators of osmotic stress and abscisic acid (ABA) signaling. Evidence presented herein indicates that CPL3 and CPL4 are homologs of a prototype CTD phosphatase, FCP1 (TFIIF-interacting CTD-phosphatase). CPL3 and CPL4 contain catalytic FCP1 homology and breast cancer 1 C terminus (BRCT) domains. Recombinant CPL3 and CPL4 interact with AtRAP74, an Arabidopsis ortholog of a FCP1-interacting TFIIF subunit. A CPL3 or CPL4 C-terminal fragment that contains the BRCT domain mediates molecular interaction with AtRAP74. Consistent with their predicted roles in transcriptional regulation, green fluorescent protein fusion proteins of CPL3, CPL4, and RAP74 all localize to the nucleus. cpl3 mutations that eliminate the BRCT or FCP1 homology domain cause ABA hyperactivation of the stress-inducible RD29a promoter, whereas RNAi suppression of CPL4 results in dwarfism and reduced seedling growth. These results indicate CPL3 and CPL4 are a paralogous pair of general transcription regulators with similar biochemical properties, but are required for the distinct developmental and environmental responses. CPL4 is necessary for normal plant growth and thus most orthologous to fungal and metazoan FCP1, whereas CPL3 is an isoform that specifically facilitates ABA signaling.

Male fertility depends on the proper development of the male gametophyte, successful pollen germination, tube growth, and delivery of the sperm cells to the ovule. Previous studies have shown that nutrients like boron, and ion gradients or currents of Ca21, H1, and K1 are critical for pollen tube growth. However, the molecular identities of transporters mediating these fluxes are mostly unknown. As a first step to integrate transport with pollen development and function, a genome-wide analysis of transporter genes expressed in the male gametophyte at four developmental stages was conducted. Approximately 1,269 genes encoding classified transporters were collected from the Arabidopsis (Arabidopsis thaliana) genome. Of 757 transporter genes expressed in pollen, 16% or 124 genes, including AHA6, CNGC18, TIP1.3, and CHX08, are specifically or preferentially expressed relative to sporophytic tissues. Some genes are highly expressed in microspores and bicellular pollen (COPT3, STP2, OPT9), while others are activated only in tricellular or mature pollen (STP11, LHT7). Analyses of entire gene families showed that a subset of genes, including those expressed in sporophytic tissues, was developmentally regulated during pollen maturation. Early and late expression patterns revealed by transcriptome analysis are supported by promoter::b-glucuronidase analyses of CHX genes and by other methods. Recent genetic studies based on a few transporters, including plasma membrane H1 pump AHA3, Ca21 pump ACA9, and K1 channel SPIK, further support the expression patterns and the inferred functions revealed by our analyses. Thus, revealing the distinct expression patterns of specific transporters and unknown polytopic proteins during microgametogenesis provides new insights for strategic mutant analyses necessary to integrate the roles of transporters and potential receptors with male gametophyte development.

Fourteen red-fleshed plum (Prunus salicina Erhr. and hybrids) and eight peach [Prunus persica (Batsch) L.] genotypes were characterized for their total phenolic and anthocyanin contents. Selected rich phenolic genotypes showed high antioxidant activity, stable colour properties and good antimicrobial activity. Results indicated positive correlations between phenolic compounds (r2 = 0.83) and antioxidant activity for both types of fruit. Colorants prepared from an anthocyanin rich plum genotype showed similar hue to that of synthetic colorant FD&C red 3 and higher stability than a commercial red grape colorant with respect to time, temperature and pH. Additionally, a selected rich phenolic plum genotype exhibited strong antimicrobial activity against Salmonella Enteritidis and Escherichia coli O157:H7. This study proposes that selection of crops high in phenolic compounds can be related to enhanced functional properties and opens the possibility of breeding fruits with targeted functional properties for the fresh produce and processing market.

RNA isolation from ripe fruit can be complicated by high concentrations ofsugar and water. These sugars interfere with RNA extraction often resulting in low RNAquality and quantities, and high water concentrations dilute the RNA, making isolationdifficult. We report a simple but novel method by which the majority of the excess sugarand water in mature fruit of tomato (Lj'copersicon esculentu,z Mill.), watermelonlCitrzillus laitahis (Thumb.) Matsum. & Nakail, and muskmelon (Cucumis inelo L) canbe easily removed from tissue before RNA extraction. This method produced qualityRNA in a shorter time than the currently accepted method for fruit tissue RNA isolationand does not require liquid nitrogen or a freeze dryer.

Objective: An experiment evaluated the effects of citrus juice on enhancing serum antioxidant status and on osteoporosis prevention in orchidectomized rats. Method: Thirty-six 1 year-old male rats were randomized to two groups: a sham control grouop (n = 9) and an orchidectomized group (n = 27). The orchidectomized group was divided into three groups of nine and assigned to one of the following treatments: orchidectomy, orchidectomy plus orange juice, and orchidectomy plus grapefruit juice. Sixty days after initiation of the study, all rats were killed, blood was collected, and serum was harvested for total antioxidant status and indices of bone formation and resorption. Femoral density and biomechanical properties were monitored. Results: Orchaidectomy decreased (P < 0.05) total antioxidant capacity, femoral density, and biomechanical properties and increased (P -< 0.05) alkaline phosphatase, acid phosphates, and urinary excretion of hydroxyproline compared with the sham control group. In contrast to orchidectomy, orchidectomy lus orange juice and orchidectomy plus grapefruit juice reversed (P < 0.05) orchidectomy induced antioxidant suppression, decreased (P < 0.05 ) alkaline phosphatase and acid phosphatase activities, moderatly restored (P = 0.07) femoral density, increased (P < 0.05) urinary excretion of hydroxyprodoline. Conclusion: The present study supports the supposition in that drinking citrus juice positively affects serum antioxidant status and bone strength.

Oxidative stress and hypogonadism are two factors linked to the increased incidence of cardiovascular disease in males. Eating fruits and vegetables is known to reduce the incidences of oxidative stress. The objective of this research was to delineate whether drinking daily squeezed orange juice (OJ) or grapefruit juice (GJ) modulates oxidative stress and antioxidant enzymes while impacting cardiovascular risk factors in hypogonad male rats. In the present study, 36 1-year-old male rats were equally divided among the following four treatments: sham (control), orchidectomized (ORX), ORX + OJ, an and ORX + GJ. After 60 days of drinking OJ or GJ, antioxidant capacity, cholesterol, and triglycerides in serum and superoxide dismutase (SOD), catalyase (CAT), cholesterol, and triglycerides in liver were evaluated. Serum antioxidant capacity and SOD and CAT activities decreased (P < .05), while serum cholesterol and liver triglycerides increased (P < .05) in the ORX group compared with the sham group. In contrast to the ORX group, drinking OJ was ineffective while drinking GJ decreased (P < .05) triglyceride concentration in liver and increased (P < .05) serum antioxidant capacity and SOD and CAT activities compared with the ORX group. In conclusion, drinking OJ or GJ prevented oxidative stress by enhancing total antioxidant capacity and elevating liver antioxidant enzymes while modulating cardiovascular risk factors.

Bioactive compounds present in grapefruit juice are known to increase the bioavailability of certain medications by acting as potent CYP 3A4 inhibitors. An efficient technique has been developed for isolation and purification of three furocoumarins. The isolated compounds have been tested for the inhibition of human CYP 1B1 isoform using specific substrates. Grapefruit juice was extracted with ethyl acetate (EtOAc) and the dried extract was loaded onto silica gel column chromatography. Further, column fractions were subjected to preparative HPLC to obtain three compounds. The purity of these compounds was analyzed by HPLC and structures were determined by NMR studies. The identified compounds, bergamottin, 6′,7′-dihydroxybergamottin (DHB), and paradisin-A, were tested for their inhibitory effects on hydroxylase and O-dealkylase activities of human cytochrome P450 isoenzymes CYP 3A4 and CYP 1B1. Paradisin-A was found to be a potent CYP 3A4 inhibitor with an IC50 of 1.2 μM followed by DHB and bergamottin. All three compounds showed a substantial inhibitory effect on CYP 3A4 below 10 μM. Inhibitory effects on CYP 1B1 exhibited a greater variation due to the specificity of substrates. Paradisin A showed an IC50 of 3.56 ± 0.12 μM for the ethoxy resorufin O-dealkylase (EROD) activity and 33.56 ± 0.72 μM for the benzyloxy resorufin (BROD). DHB and bergamottin showed considerable variations for EROD and BROD activities with an IC50 of 7.17 μM and 13.86 μM, respectively.

Areas with mild climate conditions are suitable for growing winter spinach (Spinacia olcracea L.). Successful production depends on choosing slow-bolting cultivars resistant to major diseases in each area. Field experiments with a total of 18 cultivars were conducted during 8 years in the Winter Garden region of Texas, an area known for its high white rust (Albugo occidentalis G. W. Wils) inoculum. Spinach cultivars differed widely in their resistance to both white rust and bolting, and the incidence of both traits was more severe as the season progressed. White rust infection increased linearly with average monthly minimal air temperature. Cultivars Fidalgo, Springfield, and Springer were slow bolting and are suitable for areas with no white rust incidence, which cvs. ASR-318, DMC 66-09, Fall Green, Samish, and San Juan were more white rust resistant.

Single- and double-row arrangements of a fixed population (one plant every 0.285 m2) were compared in factorial combination with two (2002) or five (2003) cultivars for effects on yield and fruit quality of bell pepper (Capsicum annual.). Arrangements for 2002 were S30, single row 0.95 m apart, plants within rows 30 cm apart; D30, 1.9 m between centers of double-row beds, double row 30 cm apart on beds, plants within rows 30 cm apart; S37.5 cm apart. Only the S30 and D30 arrangements were used in 2003 after 2002 results showed almost no differences between S30 and S37.5 or between D30 and D37.5. Choice of cultivar was more critical in Texas, where 'X3R Wizard' consistently outperformed 'King Aruth', than in Oklahoma. Single rows resulted in more full-season total marketable fruit weight than double rows in three experiments out of four, primarily as a result of an increased weight of U. S. No. 1 fruit were single rows. Average weight per marketable fruit was consistently unaffected by plant arrangement. Single rows also resulted in a greater full-season weight of sunburned fruit than double rows in two experiments out of four. Cultivar ´ plant arrangement interactions were not evident in Oklahoma and never involved full-reason marketable fruit weights at either location in either year. Given the tested population, a single-row arrangement is likely to result in increased full-season production of U.S. No. 1 bell pepper fruit compared with a double-row arrangement, despite an increased potential for sunburned fruit with single rows.

Calmodulin (CaM) influences many cellular processes by interacting with various proteins. Here, we isolated AtBAG6, an Arabidopsis CaM-binding protein that contains a central BCL-2-associated athanogene (BAG) domain. In yeast and plants, overexpression of AtBAG6 induced cell death phenotypes consistent with programmed cell death (PCD). Recombinant AtBAG6 had higher affinity for CaM in the absence of free Ca2 + than in its presence. An IQ motif (IQXXXRGXXXR, where X denotes any amino-acid) was required for Ca2 +-independent CaM complex formation and single amino-acid changes within this motif abrogated both AtBAG6-activated CaM-binding and cell death in yeast and plants. A 134-amino-acid stretch, encompassing both the IQ motif and BAG domain, was sufficient to induce cell death. Agents generating oxygen radicals, which are known to be involved in plant PCD, specifically induced the AtBAG6 transcript. Collectively, these results suggest that AtBAG6 is a stress-upregulated CaM-binding protein involved in plant PCD.

Previously, we made a chimeric Arabidopsis thaliana vacuolar transporter CAX2B [a variant of Nterminus truncated form of CAX2 (sCAX2) containing the “B” domain from CAX1] that has enhanced calcium (Ca2+)substrate specificity and lost the manganese (Mn2+) transport capability of sCAX2.Here, we demonstrate that potato (Solanum tuberosum L.) tubers expressing theCAX2Bcontain 50–65% more calcium (Ca2+) than wild-type tubers. Moreover, expression of CAX2B in potatoes did not show any significant increase of the four metals tested, particularly manganese (Mn2+). The CAX2B-expressing potatoes have normally undergone the tuber/plant/tuber cycle for three generations; the trait appeared stable through the successive generations and showed no deleterious alternations on plant growth and development. These results demonstrate the enhanced substrate specificity of CAX2B in potato. Therefore, CAX2B can be a valuable tool for Ca2+ nutrient enrichment of potatoes with reduced accumulation of undesirable metals.

Bulb color in onions (Allium cepa) is an important trait, and homogenous red coloration is desirable in red onion cultivars. The gene encoding anthocyanin synthase (ANS) is required for anthocyanin biosynthesis in onions. We have previously described three different alleles of the ANS gene. Here we report identification of the fourth allele of ANS, ANS-hl, found in a dark red doubled haploid line. ANS-hl is similar to a non-functional allele found inBrazilian yellow cultivars except that it has several point mutations and indels throughout the promoter and coding regions, none of which are predicted to inactivate enzymatic activity. F2 and backcross populations originating from the crosses between wild-type (ANS-L) allele-containing red and pink (ANS-p) allele-containing white or yellow parents show a discrete segregation ratio of 3 red to 1 light pink, indicating that the wild-type allele is completely dominant over the pink allele. In contrast, segregating populations derived from the crosses between ANS-hl allele containing red and the same white or yellow parents show a gradient of red intensity from light pink to dark red, suggesting that other genetic factors may affect expression of ANS-hl. A newly developed PCR-based marker and two previously developed markers for allelic selection of the ANS gene were used to examine allele composition in fifty-six breeding lines and commercial cultivars. Most lines are heterogeneous for the ANS gene with two or three alleles detected. The frequency of the pink allele is low in red breeding lines, but it is predominant in white and yellow lines.

All plants sense and adapt to adverse environmental conditions, however, crop plants exhibit less genetic diversity for abiotic stress tolerance than do wild relatives indicating that a genetic basis exists for stress adaptability. Model plant genetic systems and the plethora of molecular genetic resources that are currently available are greatly enhancing our ability to identify abiotic stress-responsive genetic determinants. Forward genetic screens of T-DNA mutagenized Arabidopsis thaliana populations in the genetic background of ecotypes C24RD29a-LUC and Col-0 gl1 sos3-1 were carried out to begin an exhaustive search for such determinants. The C24RD29a-LUC screens identified mutants with altered salt/osmotic stress sensitivity or mutants with altered expression of the salt/osmotic/cold/ABA-responsive RD29a gene. Also, mutations that alter the NaCl sensitivity of sos3-1 were screened for potential genetic suppressors or enhancers of salt-stress responses mediated by SOS3. In total, more than 250 000 independent insertion lines were screened and greater than 200 individual mutants that exhibited altered stress/ABA responses were recovered. Although several of these mutants have been reported, most have not yet been studied in detail. Notable examples include novel alleles of SOS1 and mutations to genes encoding the STT3a subunit of the oligosaccharyltransferase, syntaxin, RNA polymerase II CTD phosphatases, transcription factors, ABA biosynthetic enzyme, Na+ transporter HKT1, and SUMO E3 ligase. The stress-specific phenotypes of mutations to genes that are involved in many basic cellular functions provide indication of the wide range of control mechanisms in cellular homeostasis that are involved in stress adaptation.

The aim of this study was to determine whether aminoethoxyvinylglycine (AVG), an inhibitor of ethylene synthesis, would affect earliness, increase yield, and improve overall at harvest and postharvest quality of melon (Cucumis melo L. group Cantalupensis, 'Sol Real'). Field experiments were conducted during two seasons with AVG (124 g ha-1 a.i..) applied as spray or soil injected into the root zone with a single or double application between 7 d and 21 d before harvest. The AVG soil injection method increased earliness compared with AVG spray in one season. Total marketable yield increased with AVG injection but not with the AVG spray method compared with the control. Regardless of method of application, AVG did not affect fruit firmness, rind thickness, netting, or soluble solids content when measured at harvest. However, AVG spray decreased fruit size and seed cavity in one season. Similarly, AVG spray did not affect fruit quality after storage, whereas AVG soil inject injection increased fruit firmness. Overall, melon yield and fruit quality responses to preharvest AVG applications were superior for the soil inaction than the spray method.

Netted muskmelon [Cucumis melo L. (Reticulatus Group)] fruit quality (ascorbic acid, β-carotene, total free sugars, and soluble solids concentration (SSC))) is directly related to plant potassium (K) concentration during fruit growth and maturation. During reproductive development, soil K fertilization alone is often inadequate due to poor root uptake and competitive uptake inhibition from calcium and magnesium. Foliar applications of glycine-complexed K during muskmelon fruit development has been shown to improve fruit quality; however, the influence of organiz-complexed K vs. an inorganic salt form has not bee determined. This glasshouse study investigated the effects of two K sources: a glycine-complexed K (potassium metalosate, KM) and potassium chloride (KCI) (both containing 800 mg K/L) with or without a non-ionic surfactant (Silwet L-77) on melon quality. Orange-flesh muskmelon ‘Cruiser’ was grown in a glasshouse and fertilized throughout the study with soil-applied N-P-K fertilizer. Starting at 3 to 5 d after fruit set, and up to 3 to 5 da before fruit maturity at full slip, entire plants were sprayed weekly, including the fruit, with KM or KCl with or without a surfactant. Fruit from plants receiving supplemental foliar K had significantly higher K concentrations in the edible middle mesocarp fruit tissue compared to control untreated fruit. Fruit from treated plants were also firmer, both externally and internally, than those from non-treated control plants. Increased fruit tissue firmness was accompanied by higher tissue pressure potentials of K treated plants vs. control. In general, K treated fruit had significantly higher SSC, total sugars, total ascorbic acid, and β-carotene than control fruit. Fall-grown fruit generally had higher SSC, total sugars, total ascorbic acid and β-carotene concentrations than spring-grown fruit regardless of K treatment. The effects of surfactant were not consistent, but in general, addition of a surfactant tended to affect higher SSC and β-carotene concentrations.

Netted muskmelon [Cucumis melo L. (Reticulatus Group)] fruit quality (ascorbic acid, Beta-carotene, total free sugars, and soluble solids concentration (SSC)) is directly related to plant potassium (K) concentration during fruit growth and maturation. During reproductive development, soil K fertilization along is often inadequate due to poor root uptake and competitive uptake inhibition from calcium and magnesium. Foliar applications of glycine-complexed K during muskmelon fruit development has been shown to improve fruit quality, however, the influence of organic-complexed K vs. an inorganic salt form has not been determined. This glasshouse study investigated the effects of two K sources: a glycine-complexed K (potassium metalosate, KM) and potassium chloride (KCl) (both containing 800 mg K/L) with or without a non-ionic surfactant (Silwet L-77) on melon quality. Orange-flesh muskmelon ‘Cruiser’ was grown in a glasshouse and fertilized throughout the study with soil-applied N-P-K fertilizer. Starting at 3 to 5 d after fruit set, and up to 3 to 5 d before fruit maturity at full slip, entire plants were sprayed weekly, including the fruit, with KM or KCl with or without a surfactant. Fruit from plants receiving supplemental foliar K had significantly higher K concentrations in the edible middle mesocarp fruit tissue compared to control untreated fruit. Fruit from treated plants were also firmer, both externally and internally, than those from non-treated control plants. Increased fruit tissue firmness was accompanied by higher tissue pressure potentials of K treated plants vs. control. In general, K treated fruit had significantly higher SSC, total sugars, total ascorbic acid, and Beta-carotene than control fruit. Fall-grown fruit generally had higher SSC, total sugars, total ascorbic acid and Beta-carotene concentrations than spring-grown fruit regardless of K treatment. The effects of surfactant were not consistent but in general, addition of surfactant tended to affect higher SSC and Beta-carotene concentrations.

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 (∼30%) have no significant homology to sequences published so far for other plant species. Additionally, 168 “EST-unigenes” (∼20%) correspond to genes with unknown function, whereas 410 “EST-unigenes” (∼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.

Our objectives were to identify RAPD and andromonoecioius (a) markers associated with QTL for sucrose using bulked segregant analysis in an F2 population from the melon (Cucumis melo L.) cross of 'TAM Dulce' (high sucrose) × TGR1551 (low sucrose) in a greenhouse experiment, and confirm the association of RAPD and a markers with sucrose QTL in an F2 population from the different cross of 'Deltex' (high sucrose) × TGR1551in a field experiment. Continuous distributions for sucrose were observed in the F2 populations indicating quantitative inheritance for the trait. A significant positive correlation was found between sucrose and total soluble solids (TSS). Nine RAPD markers were detected to be significantly associated with QTL for sucrose in the F2 population of the 'TAM Dulce' × TGR1551 cross in the greenhouse based on simple linear regression. Five unlinked markers associated with QTL were significant in a stepwise multiple regression analysis where the full model explained 39% of the total phenotypic variation for sucrose. Three to five of the nine RAPD markers were also observed to be significantly associated with QTL for glucose, controlled by the same QTL. Four RAPD markers were confirmed in the F2 population of the 'Deltex' × TGR1551 cross in the field to be consistently associated with sucrose QTL. A significant association of a with sucrose was consistently expressed in our populations under greenhouse and field environments.

Limonoids, a family of triterpenoids with putative anticancer properties, occur in fruits as glucosides and aglycones. Both highly purified forms were isolated from seeds and molasses of citrus fruits and tested for toxic effects against two human cancer cell lines. SH-Sy5Y neuroblastoma and Caco-2 colonic adenocarcinoma, and a noncancerous mammalian epithelial Chinese hamster ovary (CHO) cells. Viability, as quantified by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium reduction and light microscopy, was shortened significantly (P < 0.001) in cancer cells exposed to aglycones, viz, limonin, nomilin, obacunone, and deacetylnomillin. SH-SY5Y cells were more sensitive than Caco-2 cells to the limonoids, whereas noncancerous CHO cells showed hardly any change in cell numbers or cell morphology. Aglycone toxicity was dose dependent, but below the killing potential of glucosides. This observation correlated with a slower rate of induction of caspase 3/7 activity by agllycones. A flow cytometric analysis of SH-SY5Y cells treated with glucosides and aglycones showed an increased ploidy, which is consistent with enhancing chromosomal abnormalities The results confirm that limonoids exert a strong multifaceted lethal action against cancer cells, but are relatively ineffective against CHO cells. Of the two, metabolites derived from glucosides are the more likely progenitors of an apoptosis response in situ.

Cell life and cell death in vivo are delicately balanced and linked processes dependent upon several “death” or “survival” signals from inside and outside the cell. These signals are regulated by several vital intracellular events associated with DNA, RNA, caspases, proteins, and ions. Drugs and other chemicals are known to threaten the stability of such key intracellular molecules by causing ion deregulation, oxidative stress, DNA fragmentation and activation of caspases. Citrus flavonoids, such as hesperidin (HES) and rutin (RUT), are known to specifically interfere with such deleterious pathways, minimize macromolecular perturbations, and exhibit their prolific antitoxic properties. This study was designed to investigate whether HES and RUT pre-exposures (1.25 g/kg orally for 14 days) have the ability to counteract hepatotoxicity and nephrotoxicity induced by toxic doses of acetaminophen (APAP, 50 mg/kg, ip) and dislofenac (DCLF, 200 mg/kg, po) respectively in vivo. Additional objectives were to determine whether exposure to these flavonoids protect the liver and kidneys from oxidative stress and genomic DNA fragmentation, the prime players responsible for turning on various forms of cell death. Results indicate that HES and RUT pre-exposures showed dramatic prevention of hepato- and nephrotoxicity by minimizing toxicant-induced oxidative stress and genomic DNA fragmentation which are instrumental in orchestrating apoptotic (programmed) and necrotic (unprogrammed) cell deaths in these organs in vivo.

Steep concentration gradients of many ions are actively maintained, with lower concentrations typically located in the cytosol, and higher concentrations in organelles and outside the cell. The vacuole is an important storage organelle for many ions. The concentration gradient of cations is established across the plant tonoplast, in part, by high-capacity cation/H+ (CAX) exchange activity. While plants may not be green yeast, analysis of CAX regulation and substrate specificity has been greatly aided by utilizing yeast as an experimental tool. The basic CAX biology in Arabidopsis has immediate relevance toward understanding the functional interplay between diverse transport processes. The long-range applied goals are to identify novel transporters and express them in crop plants in order to "mine" nutrients out of the soil and into plants. In doing so, this could boost the levels of essential nutrients in plants.

Ca2+/cation antiporter (CaCA) proteins are integral membrane proteins that transport Ca2+ or other cations using the H+ or Na+ gradient generated by primary transporters. The CAX (for CAtion eXchanger) family is one of the five families that make up the CaCA superfamily. CAX genes have been found in bacteria, Dictyostelium, fungi, plants, and lower vertebrates, but only a small number of CAXs have been functionally characterized. In this study, we explored the diversity of CAXs and their phylogenetic relationships. The results demonstrate that there are three major types of CAXs: type I (CAXs similar to Arabidopsis thaliana CAX1, found in plants, fungi, and bacteria), type II (CAXs with a long N-terminus hydrophilic region, found in fungi, Dictyostelium, and lower vertebrates), and type III (CAXs similar to Escherichia coli ChaA, found in bacteria). Some CAXs were found to have secondary structures that are different from the canonical six transmembrane (TM) domains–acidic motif-five TM domain structure. Our hylogenetic tree indicated no evidence to support the cyanobacterial origin of plant CAXs or the classification of Arabidopsis exchangers CAX7 to CAX11. For the first time, these results clearly define the CAX exchanger family and its subtypes in phylogenetic terms. The surprising diversity of CAXs demonstrates their potential range of biochemical properties and physiologic relevance.

Our objectives were to identify randomly amplified polymorphic DNA (RAPD) markers associated with quantitative trait loci (QTL) for sucrose, total soluble solids (TSS), and sucrose percentage of total sugars (SPTS) using bulked segregant analysis in an F, population from the melon (Cucumis melo L.) cross of 'TAM Dulce' (high sugars) x TGR1551 (low sugars) in a greenhouse experiment, and confirm the associations of KAPD markers with QTL for these sugar traits in an F, population from the different cross of 'Deltex' (high sugars) x TGK1551 in a field experi- ment. Continuous distributions for sucrose, TSS, and SPTS were observed in the F, populations indicating quantitative inheritance for the traits. Significant positive correlations were found between sucrose and TSS or SPTS. Nine RAPD markers were detected to be significantly associated with QTL for sucrose in the F, population of the 'TAM Dulce' x TGR1551 cross in the greenhouse based on simple linear regression. Five unlinked markers associated with QTL were significant in a stepwise multiple regression analysis where the full model explained 39% of the total phenotypic varia- tion for sucrose. Five and seven of the nine RAPD markers associated with QTL for sucrose were also observed to be significantly associated with QTL for TSS and SPTS, respectively. suggesting that in this cross three sugar traits are controlled by the same QTL. Five RAPD markers were confirmed in the F, population of the 'Deltex'x TGR1551 cross in the field to be consistently associated with QTLfor three sugar traits. Significant =sociations of andromonoccious (a) with both sucrose and TSS were consistently expressed in our populations under greenhouse and field environments. These RAPD and floral markers associated with the sugar synthesis QTL identified and confirmed here could be useful in melon breeding for improving the mature fruit sweetness.

Guava fruit extracts were analyzed for antioxidant activity measured in methanol extract (AOAM), antioxidant activity measured in dichloromethane extract (AOAD), ascorbic acid, total phenolics, and total carotenoids contents. The ABTS, DPPH, and FRAP assays were used for determining both AOAM and AOAD, whereas the ORACX was used for determining only AOAM. Averaged AOAM [mM Trolox equivalent (TE)/g fresh mass (FM)] were 31.1, 25.2, 26.1, and 21.3 as detreined by the ABTS, DPPH, FRAP, and ORAC respectively. AOAM determined by all assays were well correlated with ascorbic acid (0.61 £ r £ 0.92) but had negative correlation with total carotenoids (-0.67 £ r £ -0.81).

This study evaluated the hypothesis that untreated and irradiated grapefruit as well as the isolated citrus compounds naringin and limonin would protect against azoxymethane (AOM)-induced aberrant crypt foci (ACF) by suppressing proliferation and elevating apoptosis through anti-inflammatory activities. Male Sprague–Dawley rats (n = 100) were provided one of five diets: control (without added grapefruit components), untreated or irradiated (300 Gy, 137Cs) grapefruit pulp powder (13.7 g/kg), naringin (200 mg/kg) or limonin (200 mg/kg). Rats were injected with saline or AOM (15 mg/kg) during the third and fourth week and colons were resected (6 weeks post second injection) for evaluation of ACF, proliferation, apoptosis, and cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) protein levels. Experimental diets had no effect on the variables measured in saline-injected rats. However, in AOM-injected rats, the experimental diets suppressed (P 0.02) aberrant crypt and high multiplicity ACF (HMACF; P 0.01) formation and the proliferative index (P 0.02) compared with the control diet. Only untreated grapefruit and limonin suppressed (P 0.04) HMACF/cm and expansion (P 0.008) of the proliferative zone that occurred in the AOM-injected rats consuming the control diet. All diets elevated (P 0.05) the apoptotic index in AOM-injected rats, compared with the control diet; however, the greatest enhancement was seen with untreated grapefruit and limonin. Untreated grapefruit and limonin diets suppressed elevation of both iNOS (P 0.003) and COX-2 (P 0.032) levels observed in AOM-injected rats consuming the control diet. Although irradiated grapefruit and naringin suppressed iNOS levels in AOM-injected rats, no effect was observed with respect to COX-2 levels. Thus, lower levels of iNOS and COX-2 are associated with suppression of proliferation and upregulation of apoptosis, which may have contributed to a decrease in the number of HMACF in rats provided with untreated grapefruit and limonin. These results suggest that consumption of grapefruit or limonin may help to suppress colon cancer development.

Citrus flavonoids have been shown to possess biological activities such as anti-inflammatory properties, cholesterol lowering and immune system modulation. In this study, 12 made-from-concentrate (MFC) and 14 not-from-concentrate (NFC) orange juices, and five NFC grapefruit juices available in the US market were analyzed for their flavonoid content by reverse phase HPLC. Individual and total flavonoid content was determined for all of the brands. The correlation between flavonoid content (mg) and price (US dollar) per unit volume of orange and grapefruit juices were also evaluated. Significant differences (P0.05) among the brands and within the brand were observed for all of the prominent flavanone glucosides. Within the brand, juice types containing added antioxidant vitamins C and E were not superior in flavonoid content compared to orange juice types devoid of added antioxidant vitamins. Total flavonoid content of MFC orange juices (53 mg/100 mL; n=12) was significantly (P0.05) higher than NFC orange juices (36.5 mg/100 mL; n=14). Hesperidin was found to be the major flavonoid followed by narirutin and didymin in orange juice. Naringin, narirutin, and poncirin were the major flavonoids in all brands of grapefruit juices. The concentration of didymin was considerably higher in NFC orange juices compared to MFC orange juices. Interestingly, no correlation was observed between price and the total flavonoid content of MFC orange juices and NFC grapefruit juices. However, a significant negative correlation (r=-0.49; P=0.001) was observed for NFC orange juices. This study provides valuable information on flavonoid composition of orange and grapefruit juices commonly available in the US market.

Fruits contain a range of phenolics and carotenoids which have been implicated in improving human health. The objective of this study was to characterize the phytochemicals and antioxidant activity (AOA) exhibited in peaches and plums and to determine if any associations existed between these phytochemicals and AOA. Twenty-two peach varieties and fifty-three plum varieties with different flesh and skin color were analyzed for their antioxidant content and AOA. Total phenolics, anthocyanins, and carotenoids were analyzed spectrophotometrically. AOA was evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH). Carotenoid content was higher in yellow-flesh (2-3 mg β-carotene/100 g fw-fresh weight) than in white or red-flesh peaches (0.01-1.8 mg β-carotene/100 g fw). AOA was about 2-fold higher in red-flesh varieties than in white/yellow-flesh peach varieties. Among the peaches, the AOA was best correlated with phenolic content. Among the plums, the anthocyanin content increased with the red color intensity. Although the plums varied widely in phenolic content, the red/purple-flesh plums generally had higher phenolic content (400-500 mg chlorogenic acid/100 g fw) than the other plums. Carotenoid content in plums was similar for all varieties. AOA tended to be higher in red/purple-flesh varieties as compared to light colored flesh plums. As with the peaches, the best correlations were between the AOA and the total phenolics content of the fruit.

This study was conducted to determine the effects of genetic and environmental factors on onion pungency, estimated as pyruvic acid levels. Genetically identical clones were grown at three different field locations and in a greenhouse. Onion pungency was significantly influenced by clone type, location, and their interaction. Genetic differences were the major determining factor of onion pungency (81.3% of total variation). Location, including all environmental factors, and the clone × location interaction comprised 11.4% and 7.3% of the total variation, respectively. The magnitude of the pungency difference among field-grown onions was about 1.5 μmol/ml. The pungency levels were not positively correlated with soil sulfur nutrition levels, which ranged from 16 to 97 ppm. Within clones, onion pungency levels were loosely inversely correlated with increasing bulb weight. The clones proved to have the most uniform pungency (8% CV), followed by hybrids (10.6% CV) and open-pollinated cultivars (21.3% CV). We have demonstrated that genetic factors were determinant of onion pungency. Environmental factors influenced pungency to a lesser degree. Therefore, choosing cultivars with low pungency, ideal growing environments and proper sulfur nutrition control, are key factor in producing sweet onions.

Limonoid glucosides (primarily limonin 17--D-glucopyranoside, LG) were extracted from grapefruit molasses by supercritical fluid extraction using a supercritical carbon dioxide-ethanol (SC CO2-ethanol) system. Extraction conditions to maximize the yield of LG were determined by varying pressure, temperature, ethanol concentration, and extraction time. The highest yield of LG at 0.61 mg/g molasses was obtained at a pressure 48.3 MPa, a temperature of 50 C, 10% ethanol (XEth = 0.1), and 40 min of extraction time at a flow rate of 5.0 L/min. The results demonstrated that SC CO2 extraction of limonoid glucosides from grapefruit molasses has practical significance for commercial production.

To determine the mechanisms by which dietary fish oil (FO) affects antigen-stimulated Th1 cell development, DO11.10 Rag 2–/– T cell receptor transgenic mice were fed a control diet (5% corn oil (CO) or a FO diet (1% CO + 4% FO, (n-3) PUFA) for 2 wk. CD4+ T cells were cultured under neutral or Th1 polarizing conditions. FO feeding suppressed (P < 0.05) ovalbumin peptide–induced proliferation of nonpolarized CD4+ T cells. Differentiation in vitro to Th1 cells was not affected by dietary FO, as evidenced by similar percentages of KJ1–26+, IFN-+, IL-4– Th1 cells in cultures from CO-fed (99%) and FO-fed (97%) mice. However, the absolute number of viable Th1 cells in polarized cultures from FO-fed mice was less than half that observed in CO-fed mice (P < 0.05), indicating that FO inhibits in vitro Th1 clonal expansion. The reduced number of Th1 cells in FO cultures was not a result of increased apoptosis, because similar percentages of apoptotic Th1 cells were observed in cultures from FO- and CO-fed mice. IL-2–induced cell proliferation was significantly decreased in polarized Th1 cells from the FO group; however, the suppressed proliferation was not linked to reduced CD25 surface expression on antigen-stimulated CD4+ T cells. Adoptively transferred CFSE-labeled DO11.10 CD4+ cells into immunized mice (Th1 polarizing agents) showed that dietary FO reduced (P < 0.05) the number of cell divisions in vivo. These studies suggest that the attenuated inflammatory response which accompanies FO feeding may be explained, at least in part, by suppression of Th1 clonal expansion.