Ganesh R. Panta

Transient activation of NF-kappaB through a TAK1/IKK kinase pathway by TGF-beta1 inhibits AP-1/SMAD signaling and apoptosis: implications in liver tumor formation.

NF-κB has been implicated in the regulation of apoptosis, a key mechanism of normal and malignant growth control. Previously, we demonstrated that inhibition of NF-κB activity by TGF-β1 leads directly to induction of apoptosis of murine B-cell lymphomas and hepatocytes. Thus, we were surprised to determine that NF-κB is transiently activated in response to TGF-β1 treatment. Here we elucidate the mechanism of TGF-β1-mediated regulation of NF-κB and induction of apoptosis in epithelial cells. We report that TGF-β1 activates IKK kinase, which mediates IκB-α phosphorylation. In turn, the activation of IKK following TGF-β1 treatment is mediated by the TAK1 kinase. As a result of NF-κB activation, IκB-α mRNA and protein levels are increased leading to postrepression of NF-κB and induction of cell death. Inhibition of NF-κB following TGF-β1 treatment increased AP-1 complex transcriptional activity through sustained c-Jun phosphorylation, thereby potentiating AP-1/SMADs-mediated cell killing. Furthermore, TGF-β1-mediated upregulation of Smad7 appeared independent of NF-κB. In hepatocellular carcinomas of TGF-β1 or TGF-α/c-myc transgenic mice, we observed constitutive activation of NF-κB that led to inhibition of JNK signaling. Overall, our data illustrate an autocrine mechanism based on the ability of IKK/NF-κB/IκB-α signaling to negatively regulate NF-κB levels thereby permitting TGF-β1-induced apoptosis through AP-1 activity.

Genetic analysis of freezing tolerance in blueberry (Vaccinium section Cyanococcus)

Abstract An understanding of the genetic control of freezing tolerance (FT) in woody perennials is important for the effective selection and development of plants with a broader climatic adaptation. This study was undertaken to examine the inheritance and gene action of FT in segregating populations of a woody perennial blueberry (Vaccinium, section Cyanococcus). Two backcross populations were derived from interspecific hybrids of the diploid species Vaccinium darrowi andVaccinium caesariense, which are widely divergent in their FT. The bud FTs of uniformly cold acclimated plants of parental, F1, and two backcross populations were evaluated with a laboratory controlled freeze-thaw regime, followed by a visual assessment of injury. FT (LT50) was defined as the temperature causing 50% of the flower buds to be injured. Data indicate that the two parents were homozygous for genes for low or high FT. Freezing-tolerance values of the parental and F1 populations indicate that freeze-sensitivity is a partially dominant trait. Results from reciprocal crosses revealed that there was no significant maternal influence on freezing tolerance. Parental phenotypes were fully recovered in 40–42 plants of each testcross population, suggesting that FT is determined by relatively few genes. The degree of dominance and an analysis of generation means revealed that FT in blueberry is controlled largely by additive gene effects and, to a lesser degree, by dominance gene effects. Testing of various genetic models indicated that FT inheritance can be adequately explained by a simple additive-dominance model; however, two epistatic models involving additive-additive and dominance-dominance interactions also fit the data.

Inheritance of Cold Hardiness and Dehydrin Genes in Diploid Mapping Populations of Blueberry

Summary Studies of herbaceous plants suggest that cold hardiness is a complex, quantitatively inherited trait. Although development of cold hardiness is an integral part of the life cycle of woody perennial plants, studies on the genetic control of cold hardiness in woody perennials are scarce. A better understanding of the genetic control of cold hardiness would be valuable for developing more effective strategies to increase cold hardiness and, hence, climatic adaptation of woody perennial crops. In blueberry, three major dehydrins of 65, 60, and 14 kDa have been found to increase with cold acclimation and decrease with deac-climation. A comparison of these dehydrin levels among various blueberry cultivars and selections has revealed their level of accumulation to be closely associated with cold hardiness level. Efforts are underway to isolate and map the dehydrin genes of blueberry utilizing blueberry populations that segregate for cold hardiness in order to determine if the dehydrin genes map to or co-segregate with QTLs controlling cold hardiness. Progress has been made toward this goal. Cold hardiness levels were determined for a portion of the blueberry mapping populations (derived from testcrosses of Vaccinium darrowi Camp X V. caesariense Mackenz. F1s to another V. darrowi and another V. caesariense) using a laboratory controlled freeze-thaw regime, followed by visual assessment of injury to floral buds. As expected, the V. darrowi and V. caesariense parents were found to differ significantly in terms of cold hardiness levels (LT50s of -13°C and -20°C, respectively). Mean cold hardiness level of F1s (LT50 of -14.7°C) was skewed toward the V. darrowi parents suggesting that cold hardiness is a partially recessive trait. The sequence of a 2.0 kb cDNA clone, which encodes the 60 kDa blueberry dehydrin, was used to map a dehydrin-related gene to current linkage group 12 of the V. caesariense testcross population. A preliminary comparison of the segregation pattern of the dehydrin-related gene to that of the cold hardiness trait suggests that the marker does not segregate with cold hardiness.

Effect of cold and drought stress on blueberry dehydrin accumulation

Summary Dehydrins are a group of plant proteins which respond to any type of stress that causes dehydration at the cellular level, such as cold and drought stress. Previously, three dehydrins of 65, 60, and 14.kDa were identified as the predominant proteins present in cold acclimated blueberry (Vaccinium corymbosumLinn.) floral buds. Levels were shown to increase with cold acclimation and decrease with deacclimation and resumption of growth. In the present study, to determine if dehydrins are induced in other organs in response to low temperature treatment (48C) and in response to drought, accumulation of dehydrins was examined in leaves, stems, and roots of two cultivars and one wild selection (a V. corymbosum cultivar, a V. ashei Reade cultivar, and a V. darrowi Camp selection) of blueberry by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting. Cold treatment involved placing plants in a cold room maintained at 48C for five weeks; drought stress was imposed by withholding water from potted, greenhouse-grown plants for 34.d. Relative water content (RWC) of shoots was determined periodically throughout the drought treatment. Dehydrins accumulated with both cold and drought stress but their molecular masses varied depending upon blueberry species. Dehydrins accumulated to higher levels in stems and roots than in leaves with cold stress and to higher levels in stems than in either roots or leaves with drought stress. Furthermore, cold treatment combined with dark treatment induced higher levels of dehydrins than cold treatment combined with a 10.h light/14.h dark photoperiod, suggesting that dehydrins may be responsive to changes in photoperiod as well. In the cold-stress experiment, the level of dehydrin accumulation was correlated with expected level of plant cold hardiness in the three genotypes. In the drought stress experiment, dehydrins accumulated prior to significant changes in RWC, and dehydrin levels did not appear to be closely correlated with RWC either among or within genotypes.

Genetic Control of Cold Hardiness in Blueberry

Winter hardiness in woody perennials depends on the complex integration of two phenological events: endodormancy and development of cold hardiness (cold acclimation; CA) (Powell 1987). Exposure to low temperatures during fall and winter, which plays a role in CA, is also required for breaking endodormancy and resumption of growth the following spring (Scalabrelli and Couvillion 1986). This requirement, called chilling requirement (CR), is genetically determined (Hauagge and Cummins, 1991, Samish, 1954). Although limited in number, most studies indicate that cold hardiness (CH) is a complex trait with polygenic inheritance. Despite their integral role in the life cycle of woody perennials, studies aimed at elucidating genetic control of CH and CR are scarce.