Rachel M. Hackett

Identification of a Specific Isoform of Tomato Lipoxygenase (TomloxC) Involved in the Generation of Fatty Acid-Derived Flavor Compounds

There are at least five lipoxygenases (TomloxA, TomloxB, TomloxC, TomloxD, and TomloxE) present in tomato (Lycopersicon esculentum Mill.) fruit, but their role in generation of fruit flavor volatiles has been unclear. To assess the physiological role of TomloxC in the generation of volatile C6 aldehyde and alcohol flavor compounds, we produced transgenic tomato plants with greatly reduced TomloxC using sense and antisense constructs under control of the cauliflower mosaic virus 35S promoter. The expression level of the TomloxC mRNA in some transgenic plants was selectively reduced by gene silencing or antisense inhibition to between 1% and 5% of the wild-type controls, but the expression levels of mRNAs for the four other isoforms were unaffected. The specific depletion of TomloxC in transgenic tomatoes led to a marked reduction in the levels of known flavor volatiles, including hexanal, hexenal, and hexenol, to as little as 1.5% of those of wild-type controls following maceration of ripening fruit. Addition of linoleic or linolenic acid to fruit homogenates significantly increased the levels of flavor volatiles, but the increase with the TomloxC-depleted transgenic fruit extracts was much lower than with the wild-type control. Confocal imaging of tobacco (Nicotiana tabacum) leaf cells expressing a TomloxC-GFP fusion confirmed a chloroplast localization of the protein. Together, these results suggest that TomloxC is a chloroplast-targeted lipoxygenase isoform that can use both linoleic and linolenic acids as substrates to generate volatile C6 flavor compounds. The roles of the other lipoxygenase isoforms are discussed.

hydra Mutants of Arabidopsis Are Defective in Sterol Profiles and Auxin and Ethylene Signaling

The hydra mutants of Arabidopsis are characterized by a pleiotropic phenotype that shows defective embryonic and seedling cell patterning, morphogenesis, and root growth. We demonstrate that the HYDRA1 gene encodes a Δ8-Δ7 sterol isomerase, whereas HYDRA2 encodes a sterol C14 reductase, previously identified as the FACKEL gene product. Seedlings mutant for each gene are similarly defective in the concentrations of the three major Arabidopsis sterols. Promoter::reporter gene analysis showed misexpression of the auxin-regulated DR5 and ACS1 promoters and of the epidermal cell file–specific GL2 promoter in the mutants. The mutants exhibit enhanced responses to auxin. The phenotypes can be rescued partially by inhibition of auxin and ethylene signaling but not by exogenous sterols or brassinosteroids. We propose a model in which correct sterol profiles are required for regulated auxin and ethylene signaling through effects on membrane function.

Cloning and characterization of tomato leaf senescence-related cDNAs

Senescence-related cDNA clones designated SENU1, 4, 5 (senescence up-regulated) and SEND32, 33, 34, 35 and 36 (senescence down-regulated) isolated from a tomato leaf cDNA library [9] were characterized. Southern analysis showed that SEND32 is encoded by a single-copy gene while SEND33, 34, 35, 36 and SENU1 and SENU5 are members of small gene families. DNA and protein database searches revealed that SEND32, SEND35, SENU1 and SENU5 are novel cDNAs of unknown function. SEND33 encodes ferredoxin, SEND34 encodes a photosystem II 10 kDa polypeptide and SEND36 encodes catalase. The SENU4 sequence is identical to the P6 tomato protein previously reported to be pathogenesis-related [46]. The mRNA levels of SENU1, 4 and 5 increased during leaf senescence and SENU1 and SENU5 were also expressed at high levels during leaf development and in other plant organs. The SENU4 mRNA was associated more specifically with leaf senescence, although low expression was also detected in green fruit. The mRNAs for all SEND clones decreased during tomato leaf development and senescence and all except SEND32 were expressed at low levels in other plant organs. The accumulation of mRNA homologous to SENU4 and the decrease in abundance of SEND32 provide good molecular markers for leaf senescence.

A Simple, Reliable Tool for Owners to Assess the Body Condition of Their Dog or Cat

Numerous methods exist for quantifying body composition and body fat mass in companion animals. In a clinical setting, the most widely accepted and practical method of body condition evaluation is condition scoring using visual assessment and palpation (1). All such systems attempt to partition a body composition continuum into a finite number of categories. Currently, 3 main systems exist, all of which use similar visual and palpable characteristics, but which differ by the number of integer categories within the scoring system (e.g., 5 points, 6 points, and 9 points) (2–8). The most widely accepted system is the 9-integer scale system, which has previously been shown to correlate well with body fat mass determined by dual-energy X-ray absorptiometry (DXA) (2–4). To aid decision making, a series of animal silhouettes are also provided that illustrate the visual characteristics for a typical (e.g., Labrador morphology) dog and cat. Scores determined by different operators have also been shown to correlate well (2–4), although a degree of expertise is required, rendering this system less accessible to untrained pet owners. S.H.A.P.E (Size, Health And Physical Evaluation) is a new algorithm-based system that uses similar visual and palpable characteristics as existing scoring systems (see http://www.petslimmers.com/shape.htm). A series of questions are followed that direct the operator to examine the animal in a sequential fashion. The questions instruct the operator to perform examinations that will determine the presence and amount of subcutaneous fat (over the ribcage and spine, etc.), and the amount of abdominal fat (by determining the presence and degree of abdominal tuck). Ultimately, 1 of 7 categories of body condition is chosen, each of which is assigned an alphabetical character from A (underweight) to G (obese). Letters were chosen for this new system to avoid confusion with current body condition score systems. This approach is designed to minimize interoperator variability and expertise required, allowing owners to evaluate their animals in the home and consult the veterinarian accordingly. The aim of the current study was to assess the performance of the algorithm system in predicting body composition in dogs and cats, and to acquire preliminary data on how the system performed in the hands of both experienced and inexperienced operators.

LeCTR2, a CTR1-like protein kinase from tomato, plays a role in ethylene signalling, development and defence.

Arabidopsis AtCTR1 is a Raf-like protein kinase that interacts with ETR1 and ERS and negatively regulates ethylene responses. In tomato, several CTR1-like proteins could perform this role. We have characterized LeCTR2, which has similarity to AtCTR1 and also to EDR1, a CTR1-like Arabidopsis protein involved in defence and stress responses. Protein–protein interactions between LeCTR2 and six tomato ethylene receptors indicated that LeCTR2 interacts preferentially with the subfamily I ETR1-type ethylene receptors LeETR1 and LeETR2, but not the NR receptor or the subfamily II receptors LeETR4, LeETR5 and LeETR6. The C-terminus of LeCTR2 possesses serine/threonine kinase activity and is capable of auto-phosphorylation and phosphorylation of myelin basic protein in vitro. Overexpression of the LeCTR2 N-terminus in tomato resulted in altered growth habit, including reduced stature, loss of apical dominance, highly branched inflorescences and fruit trusses, indeterminate shoots in place of determinate flowers, and prolific adventitious shoot development from the rachis or rachillae of the leaves. Expression of the ethylene-responsive genes E4 and chitinase B was upregulated in transgenic plants, but ethylene production and the level of mRNA for the ethylene biosynthetic gene ACO1 was unaffected. The leaves and fruit of transgenic plants also displayed enhanced susceptibility to infection by the fungal pathogen Botrytis cinerea, which was associated with much stronger induction of pathogenesis-related genes such as PR1b1 and chitinase B compared with the wild-type. The results suggest that LeCTR2 plays a role in ethylene signalling, development and defence, probably through its interactions with the ETR1-type ethylene receptors of subfamily I.

SlTPR1, a tomato tetratricopeptide repeat protein, interacts with the ethylene receptors NR and LeETR1, modulating ethylene and auxin responses and development

The gaseous hormone ethylene is perceived by a family of ethylene receptors which interact with the Raf-like kinase CTR1. SlTPR1 encodes a novel TPR (tetratricopeptide repeat) protein from tomato that interacts with the ethylene receptors NR and LeETR1 in yeast two-hybrid and in vitro protein interaction assays. SlTPR1 protein with a GFP fluorescent tag was localized in the plasmalemma and nuclear membrane in Arabidopsis, and SlTPR1-CFP and NR-YFP fusion proteins were co-localized in the plasmalemma and nuclear membrane following co-bombardment of onion cells. Overexpression of SlTPR1 in tomato resulted in ethylene-related pleiotropic effects including reduced stature, delayed and reduced production of inflorescences, abnormal and infertile flowers with degenerate styles and pollen, epinasty, reduced apical dominance, inhibition of abscission, altered leaf morphology, and parthenocarpic fruit. Similar phenotypes were seen in Arabidopsis overexpressing SlTPR1. SlTPR1 overexpression did not increase ethylene production but caused enhanced accumulation of mRNA from the ethylene responsive gene ChitB and the auxin-responsive gene SlSAUR1-like, and reduced expression of the auxin early responsive gene LeIAA9, which is known to be inhibited by ethylene and to be associated with parthenocarpy. Cuttings from the SlTPR1-overexpressors produced fewer adventitious roots and were less responsive to indole butyric acid. It is suggested that SlTPR1 overexpression enhances a subset of ethylene and auxin responses by interacting with specific ethylene receptors. SlTPR1 shares features with human TTC1, which interacts with heterotrimeric G-proteins and Ras, and competes with Raf-1 for Ras binding. Models for SlTPR1 action are proposed involving modulation of ethylene signalling or receptor levels.

Genetic mapping of ripening and ethylene-related loci in tomato

Abstract The regulation of tomato fruit development and ripening is influenced by a large number of loci as demonstrated by the number of existing non-allelic fruit development mutations and a multitude of genes showing ripening-related expression patterns. Furthermore, analysis of transgenic and naturally occurring tomato mutants confirms the pivotal role of the gaseous hormone ethylene in the regulation of climacteric ripening. Here we report RFLP mapping of 32 independent tomato loci corresponding to genes known or hypothesized to influence fruit ripening and/or ethylene response. Mapped ethylene-response sequences fall into the categories of genes involved in either hormone biosynthesis or perception, while additional ripening-related genes include those involved in cell-wall metabolism and pigment biosynthesis. The placement of ripening and ethylene-response loci on the tomato RFLP map will facilitate both the identification and exclusion of candidate gene sequences corresponding to identified single gene and quantitative trait loci contributing to fruit development and ethylene response.

Energy Intake in Cats as Affected by Alterations in Diet Energy Density

Whether mammals have the ability to regulate total intake or intake of specific macronutrients is controversial. The definition of an animal that can regulate is one that will alter its ingestive behavior to maintain (‘‘defend’’) adequate nutrient intake (1). Locusts, for example, alter the volume ingested to maintain intake of protein and carbohydrate in the face of dilution of foods with nondigestible bulk, a range of macronutrient combinations, or variation in the frequency of, and differing distances between macronutrients (1). Another striking example comes from the work of Theall et al. (2). In those experiments, rats ingested amounts of protein and carbohydrate within a tight range when challenged with foods containing 8 different macronutrient combinations. Rapid compensation for dietary dilution was also observed in many omnivorous and herbivorous species such as chickens (3), horses (4), cows (5), and humans (6). Anecdotally, it was believed that cats were also able to regulate intake to maintain adequate macronutrient and energy intake. Small-scale studies suggested that cats consuming feed ad libitum will eventually reach a stable energy intake, but the weight at which this occurs is not predictable (7). This has important implications for feline health because the incidence of obesity is estimated at;25% (8) and is predicted to rise. If cats merely regulate to the volume eaten, then a potential solution to obesity is the production of less energy-dense foods. If cats regulate to energy or macronutrient target, however, this approach will be unsuccessful. Previous studies were conducted over short periods and without adequate power, making it difficult to draw firm conclusions (9–12). The aim of the current study was to examine the effect of dietary dilution on intake in a large population of cats over an extended period of time.