Mary A. O’Connell

Transcripts for possible capsaicinoid biosynthetic genes are differentially accumulated in pungent and non-pungent Capsicum spp

Capsaicinoids, the alkaloids responsible for pungency in chile fruit, are synthesized from phenylpropanoid intermediates and short-chain branched-fatty acids. Transcript levels of enzymes on the capsaicinoid pathway were monitored in Capsicum annuum and C. chinense fruit as a function of development, tissue type and genotype. Clones for Pal, Ca4h, and Comt were isolated from a cDNA library of habanero (C. chinense) placenta. These cDNA clones were used to measure transcript levels in different fruit tissues throughout development in six cultivars differing in pungency. Transcript levels for all three genes were positively correlated with degree of pungency in placental tissue; habanero, the most pungent chile fruit, had the highest transcript levels, CalWonder, a non-pungent fruit, had the lowest levels. Using the transcript accumulation pattern of the phenylpropanoid genes as a screening criterion, other cDNA clones have been selected. Clones for an aminotransferase, predicted to synthesize vanillylamine, and for a 3-keto-acyl ACP synthase, predicted to elongate branched-chain fatty acids, were identified. These genes are expressed in a placental-specific manner, and transcript levels are positively correlated with fruit pungency.

Chemotypic Variation of Essential Oils in the Medicinal Plant, Anemopsis californica

Anemopsis californica (Saururaceae) commonly called yerba mansa, is an important medicinal plant in many deserts in the southwestern region of North America. Populations of A. californica, collected throughout New Mexico, were examined for chemical variability in roots and rhizomes for select monocyclic (cymene, limonene, piperitone and thymol) and bicyclic (alpha-pinene, 1,8-cineole and myrtenol) monoterpenoid and phenylpropanoid (methyleugenol, isoeugenol and elemicin) derived essential oil components. Three distinct chemotypes were detected using a hierarchical clustering analysis on the concentration of 10 different analytes in three individuals from each of 17 populations. One chemotype was characterized by high elemicin concentrations, a second chemotype by high methyleugenol concentrations and the third by high piperitone and thymol concentrations. Steam distilled oil was used to screen for anticancer bioactivity. A. californica root oils demonstrated anti-proliferative activity against AN3CA and HeLa cells in vitro but no activity against lung, breast, prostate or colon cancer cells. The IC(50) values for the root oil were 0.056% and 0.052% (v/v) for the AN3CA and HeLa cells, respectively.

Carotenoid accumulation in orange-pigmented Capsicum annuum fruit, regulated at multiple levels

The pericarp of Capsicum fruit is a rich dietary source of carotenoids. Accumulation of these compounds may be controlled, in part, by gene transcription of biosynthetic enzymes. The carotenoid composition in a number of orange-coloured C. annuum cultivars was determined using HPLC and compared with transcript abundances for four carotenogenic enzymes, Psy, LcyB, CrtZ-2, and Ccs determined by qRT-PCR. There were unique carotenoid profiles as well as distinct patterns of transcription of carotenogenic enzymes within the seven orange-coloured cultivars. In one cultivar, ‘Fogo’, carrying the mutant ccs-3 allele, transcripts were detected for this gene, but no CCS protein accumulated. The premature stop termination in ccs-3 prevented expression of the biosynthetic activity to synthesize the capsanthin and capsorubin forms of carotenoids. In two other orange-coloured cultivars, ‘Orange Grande’ and ‘Oriole’, both with wild-type versions of all four carotenogenic enzymes, no transcripts for Ccs were detected and no red pigments accumulated. Finally, in a third case, the orange-coloured cultivar, Canary, transcripts for all four of the wild-type carotenogenic enzymes were readily detected yet no CCS protein appeared to accumulate and no red carotenoids were synthesized. In the past, mutations in Psy and Ccs have been identified as the loci controlling colour in the fruit. Now there is evidence that a non-structural gene may control colour development in Capsicum.

Fruit specific variability in capsaicinoid accumulation and transcription of structural and regulatory genes in Capsicum fruit.

Accumulation of capsaicinoids in the placental tissue of ripening chile (Capsicum spp.) fruit follows the coordinated expression of multiple biosynthetic enzymes producing the substrates for capsaicin synthase. Transcription factors are likely agents to regulate expression of these biosynthetic genes. Placental RNAs from habanero fruit (Capsicum chinense) were screened for expression of candidate transcription factors; with two candidate genes identified, both in the ERF family of transcription factors. Characterization of these transcription factors, Erf and Jerf, in nine chile cultivars with distinct capsaicinoid contents demonstrated a correlation of expression with pungency. Amino acid variants were observed in both ERF and JERF from different chile cultivars; none of these changes involved the DNA binding domains. Little to no transcription of Erf was detected in non-pungent Capsium annuum or C. chinense mutants. This correlation was characterized at an individual fruit level in a set of jalapeño (C. annuum) lines again with distinct and variable capsaicinoid contents. Both Erf and Jerf are expressed early in fruit development, 16-20 days post-anthesis, at times prior to the accumulation of capsaicinoids in the placental tissues. These data support the hypothesis that these two members of the complex ERF family participate in regulation of the pungency phenotype in chile.

Heat, Color, and Flavor Compounds in Capsicum Fruit

Peppers are one of the oldest vegetables known and are popular in worldwide cuisine. Members of the Capsicum genus could be considered accomplished chemists, as these plants synthesize a variety of phytochemicals with valued roles in human health and nutrition as well as sensory perception. For thousands of years, humans have selected peppers for traits that affect heat, color, and flavor in the fruit. Heat in pepper fruit is attributed to production of capsaicinoids in the placenta; this alkaloid binds to nociceptive pain receptors in mammals. Color in pepper fruit is due to a combination of pigments: chlorophylls, carotenoids, and anthocyanins accumulating in the fruit wall or pericarp of the fruit resulting in green, yellow, or purple fruit at physiological immature stages and yellow, red, or orange fruit at mature stages. Flavor is the least characterized chemistry, but is in part the result of monoterpenoids and aliphatic aldehydes, which also accumulate in the fruit wall. The synthesis and accumulation of all these phytochemicals are dependent on the genetic background of the pepper, the developmental stage of the fruit, and environmental factors.

RFLP analysis of mitochondrial DNA in two cytoplasmic male sterility systems (CMS-D2 and CMS-D8) of cotton

Cytoplasmic male sterility (CMS) in higher plants is a maternally inherited trait and CMS-associated genes are known to be located in the mitochondrial genome. However, CMS-inducing genes in CMS-D2 and CMS-D8 of Upland cotton (Gossypium hirsutum L., AD1) are currently unknown. The objective of this study was to identify potential candidate DNA or gene sequences for CMS-D2 and CMS-D8 through restriction fragment length polymorphism (RFLP) analysis. Seven mtDNA gene probes and five restriction enzymes were first used to compare D2 (from G. harknessii Brandegee) and AD1 cytoplasms. With cox1, cox2, and atp1 as probes, RFLP polymorphisms were detected with one or more restriction enzyme digestions. The most notable difference was an additional fragment in the normal AD1 cytoplasm detected by cox2 in digests of three enzymes, and by cox1 and atp1 in digests with PstI. The RFLP analysis was then conducted among CMS-D2, CMS-D8 (from G. trilobum (DC.) Skovst.), and AD1 cytoplasms. Two probes from maize, atp1 and atp6, detected polymorphism among the different cytoplasmic lines. However, no difference in RFLP patterns was noted between male sterile (A) and restorer (R) lines with the D2 or D8 cytoplasm, indicating that the presence of the D2 or D8 restorer gene does not affect mtDNA organization in Upland cotton. The results demonstrate that RFLP using atp1 and atp6 as probes can distinguish the three cytoplasms. The atp1 and atp6 in CMS-D8 and these two genes together with cox1 and cox2 in CMS-D2 could be the candidates of CMS-associated genes in the mitochondrial genome, providing information for further molecular studies and developing PCR-based markers for the CMS cytoplasms in breeding. This research represents the first work using RFLP to analyze the genetic basis of CMS in cotton.

Dinoxin B, a Withanolide from Datura inoxia Leaves with Specific Cytotoxic Activities

A new withanolide, dinoxin B (12,21-dihydroxy-1-oxowitha-2,5,24-trienolide-27-O-β-D-glucopyranoside, 1), was isolated from a methanol extract of Datura inoxia leaves, using bioassay-guided fractionation. The structure was determined by spectroscopic techniques, including (1)H, (13)C, and 2D NMR experiments as well as by HRMS. Extracts and the purified compound were tested for their antiproliferative activities toward a panel of human normal and cancer cell lines. Dinoxin B (1) and its aglycone (2) exhibited submicromolar IC(50) values against multiple human cancer cell lines. Among the most sensitive were several breast cancer cell lines. Dinoxin B (1) was found only in D. inoxia and was not detected in D. metel or D. stramonium. The accumulation of this compound was limited largely to leaf tissue, with little to none detected in extracts from the flowers, fruits, roots, or stems of D. inoxia.

Assessing Colorectal Cancer Screening Behaviors and Knowledge among At-Risk Hispanics in Southern New Mexico

Purpose Colorectal cancer (CRC) mortality rates in New Mexico (NM) continue to be higher than national rates. Hispanic CRC mortality rates in NM surpass those of overall Hispanics in the US. This study was designed to characterize and understand factors contributing to low CRC screening rates in this border region. Methods A CRC Knowledge Assessment Survey (KAS) was administered in either English or Spanish to 247 individuals attending community events throughout southern NM. A subset of these individuals completed an online CRC risk assessment survey managed by the National Cancer Institute (NCI). Data analysis tested for significant differences in knowledge, physician-patient CRC interactions, CRC risk level perception, and screening rates across diverse ethnic and age groups. Results Both CRC knowledge and physician-patient CRC interactions were positively associated with participant screening history. Significant age and ethnic differences for CRC knowledge, physician-patient CRC interactions, and screening history in the NM border sample were also seen. Age-eligible Hispanics (50+) as well as those less than 50 years of age had lower CRC knowledge and were less likely to engage in physician-patient CRC interactions than non-Hispanic Whites (NHWs). The age-eligible Hispanics also reported lower CRC screening rates than their NHW counterparts. Conclusions Low CRC knowledge and limited physician-patient CRC interactions appear to contribute to low screening rates in this NM population. Expanding education and outreach efforts for this border population are essential to promote early CRC detection and thereby decrease overall CRC mortality rates.

Correlations of carotenoid content and transcript abundances for fibrillin and carotenogenic enzymes in Capsicum annum fruit pericarp.

The fruits of Capsicum spp. are especially rich sites for carotenoid synthesis and accumulation, with cultivar-specific carotenoid accumulation profiles. Differences in chromoplast structure as well as carotenoid biosynthesis are correlated with distinct carotenoid accumulations and fruit color. In the present study, the inheritance of chromoplast shape, carotenoid accumulation profiles, and transcript levels of four genes were measured. Comparisons of these traits were conducted using fruit from contrasting variants, Costeño Amarillo versus Costeño Red, and from F1 hybrids; crosses between parental lines with novel versions of these traits. Intermediate chromoplast shapes were observed in the F1, but no association between specific carotenoid accumulation and chromoplast shape was detected. Increased total carotenoid content was associated with increased β-carotene and violaxanthin content. Transcript levels for phytoene synthase (Psy) and β-carotene hydroxylase (CrtZ-2) were positively correlated with increased levels of specific carotenoids. No correlation was detected between transcript levels of capsanthin/capsorubin synthase (Ccs) and carotenoid composition or chromoplast shape. Transcript levels of fibrillin, were differentially correlated with specific carotenoids, negatively correlated with accumulation of capsanthin, and positively correlated with violaxanthin. The regulation of carotenoid accumulation in chromoplasts in Capsicum fruit continues to be a complex process with multiple steps for control.

Somatic Hybridization in Tomato

The reasons for subjecting the tomato eultivar, Lycopersicon esculentum, to numerous cell and tissue culture studies have been twofold: agronomic improvement of this important crop species and exploitation of tomatO’s abundant classical genetics for basic research purposes. Several years ago we began experiments utilizing protoplasts of Lycopersicon species, with the goals of developing methods to culture and regenerate plants, influencing transmission of genomes following cell fusion, and genetically analyzing plants resulting from protoplast or fusion product regeneration. At the time, little information was available about the requirements for successful culture and regeneration of members of the tomato genus. We looked to the facile protoplast culture and fusion of other Solanaceous species, Nicotiana and Petunia, as guides, as well as the arduous but effective methods for potato protoplasts developed by Shepard et al. (1980). Here we will summarize our knowledge of Lycopersicon protoplast culture and fusion and the analysis of regenerated plants.