Seasonal shifts in dormancy status, carbohydrate metabolism, and related gene expression in crown buds of leafy spurge

Crown buds of field-grown leafy spurge (Euphorbia esula L.) were examined to determine relationships between carbohydrate metabolism and gene expression throughout para-, endo-, and eco-dormancy during the transition from summer, autumn, and winter, respectively. The data indicates that endo-dormancy plays a role in preventing new shoot growth during the transition from autumn to winter. Cold temperature was involved in breaking endo-dormancy, inducing flowering competence, and inhibiting shoot growth. An inverse relationship developed between starch and soluble sugar (mainly sucrose) content in buds during the shift from para- to endo-dormancy, which continued through eco-dormancy. Unlike starch content, soluble sugars were lowest in crown buds during para-dormancy but increased over two- to three-fold during the transition to endo-dormancy. Several genes (AGPase, HK, SPS, SuSy, and UGPase) coding for proteins involved in sugar metabolism were differentially regulated in conjunction with well-defined phases of dormancy in crown buds. Marker genes for S-phase progression, cell wall biochemistry, or responsive to auxin were also differentially regulated during transition from para-, endo-, and eco-dormancy. The results were used to develop a model showing potential signalling pathways involved in regulating seasonal dormancy status in leafy spurge crown buds.     

Molecular analysis of signals controlling dormancy and growth in underground adventitious buds of leafy spurge

Dormancy and subsequent regrowth of adventitious buds is a critical physiological process for many perennial plants. We have used the expression of hormone and cell cycle-responsive genes as markers to follow this process in leafy spurge (Euphorbia esula). In conjunction with earlier studies, we show that loss of mature leaves results in decreased sugar levels and increased gibberellin perception in underground adventitious buds. Gibberellin is sufficient for induction of S phase-specific but not M phase-specific gene expression. Loss of both apical and axillary buds or inhibition of polar auxin transport did not result in induction of S phase- or M phase-specific gene expression. Loss of polar auxin transport was necessary for continuation of the cell cycle and further bud development if the S phase was previously initiated.     

Potential roles for autophosphorylation,kinase activity,and abundance of a CDK-activating kinase (Ee;CDKF;1) during growth in leafy spurge

Leafy spurge (Euphorbia esula L.) is a deep-rooted perennial weed that propagates both by seeds and underground adventitious buds located on the crown and roots. To enhance our understanding of growth and development during seed germination and vegetative propagation, a leafy spurge gene (Accession No. AF230740) encoding a CDK-activating kinase (Ee;CDKF;1) involved in cell-cycle progression was identified, and its function was confirmed based on its ability to rescue a yeast temperature-sensitive CAK mutant (GF2351) and through in vitro kinase assays. Site-directed mutagenesis of Ee;CDKF;1 indicated that two threonine residues (Thr291 and Thr296) were mutually responsible for intra-molecular autophosphorylation and for phosphorylating its substrate protein, cyclin-dependent kinase (CDK). Polyclonal antibodies generated against the Ee;CDKF;1 protein or against a phosphorylated Ee;CDKF;1 peptide [NERYGSL(pT)SC] were used to examine abundance and phosphorylation of CDKF;1 during seed germination and bud growth. The levels of CDKF;1 were lower in dry or imbibed seeds than in germinating seeds or seedlings. Differences in CDKF;1 were also observed during adventitious bud development; small buds appeared to have greater levels of CDKF;1 than large buds. Similar patterns of CDKF;1 expression were detected with either the polyclonal antibody developed using the CDKF;1 protein or the phosphorylated peptide. These results indicated that Thr291 is constitutively phosphorylated in vivo and associated with Ee;CDKF;1 activity. Our results further suggest that a certain level of CDKF;1 activity is maintained in most tissues and may be an important phenomenon for enzymes that regulate early steps in cell-cycle signaling pathways. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

The cross-talk of NOTCH and GSK-3 signaling in colon and other cancers

The GSK-3 kinases, GSK-3α and GSK-3β, have a central role in regulating multiple cellular processes such as glycogen synthesis, insulin signaling, cell proliferation and apoptosis. GSK-3β is the most well studied, and was originally described for its role in regulating glycogen synthase. GSK-3β has been studied as a participant in the oncogenic process in a variety of cancers due to its intersection with the PTEN/PI3K/AKT and RAS/RAF/MEK/ERK pathways. Dysregulated signaling through the Notch family of receptors can also promote oncogenesis. Normal Notch receptor signaling regulates cell fate determination in stem cell pools. GSK-3β and Notch share similar targets such β-catenin and the WNT pathway. WNT and β-catenin are involved in several oncogenic processes including those of the colon. In addition, GSK-3β may directly regulate aspects of Notch signaling. This review describes how crosstalk between GSK-3β and Notch can promote oncogenesis, using colon cancer as the primary example.     

Characterization of three VERNALIZATION INSENSITIVE3-like (VIL) homologs in wild wheat, Aegilops tauschii Coss

Control of flowering time is an adaptive trait of plants for different growth habitats. A vernalization requirement is a major genetic component determining wheat flowering time. Arabidopsis VERNALIZATION INSENSITIVE3 (VIN3) and VIN3-like 1 (VIL1) play critical roles in the vernalization pathway of flowering, and three wheat VIL homologs are upregulated by vernalization in einkorn wheat. To study the relationship between vernalization and wheat VIL homologs in Aegilops tauschii, the D-genome progenitor of common wheat, we isolated three cDNAs orthologous to the einkorn wheat VIL genes. The three Ae. tauschii VIL genes showed many single nucleotide polymorphisms including non-synonymous substitutions relative to the einkorn orthologs. In addition, high rates of non-synonymous and synonymous substitutions were revealed by intraspecific variation analysis of the AetVIL sequences, suggesting adaptive evolution at the AetVIL loci. Quantitative RT-PCR analysis was conducted to examine the time course of expression of the VIL genes during vernalization. Of the three AetVIL genes, AetVIL2 was upregulated after one week of low-temperature treatment, and its expression pattern was distinct for winter and spring habit accessions. These observations strongly suggest that AetVIL2 is associated with the vernalization-responsive pathway in Ae. tauschii.     

Cloning the tomato curl3 gene highlights the putative dual role of the leucine-rich repeat receptor kinase tBRI1/SR160 in plant steroid hormone and peptide hormone signaling

Brassinosteroids (BRs) are plant steroid hormones that are essential for normal plant development. To gain better understanding of the conservation of BR signaling, the partially BR-insensitive tomato mutant altered brassinolide sensitivity1 (abs1) was identified and found to be a weak allele at the curl3 (cu3) locus. BR content is increased in both of these mutants and is associated with increased expression of DWARF: The tomato homolog of the Arabidopsis Brassinosteroid Insensitive1 Leu-rich repeat (LRR) receptor-like kinase, named tBri1, was isolated using degenerate primers. Sequence analysis of tBRI1 in the mutants cu3 and abs1 revealed that cu3 is a nonsense mutant and that abs1 is a missense mutant. A comparison of BRI1 homolog sequences highlights conserved features of BRI1 sequences, with the LRRs in close proximity to the island domain showing more conservation than N-terminal LRRs. The most homologous sequences were found in the kinase and transmembrane regions. tBRI1 (SR160) also has been isolated as the putative receptor for systemin, a plant peptide hormone. This finding suggests a possible dual role for tBRI1 in steroid hormone and peptide hormone signaling.     

The involvement of Gab1 and PI 3-kinase in beta1 integrin signaling in keratinocytes

The control of the stem cell compartment in epidermis is closely linked to the regulation of keratinocyte proliferation and differentiation. Beta1 integrins are expressed 2-fold higher by stem cells than transit-amplifying cells. Signaling from these beta1 integrins is critical for the regulation of the epidermal stem cell compartment. To clarify the functional relevance of this differential expression of beta1 integrins, we established HaCaT cells with high beta1 integrin expression by repeated flow cytometric sorting of this population from the parental cell line. In these obtained cells expressing beta1 integrins by 5-fold, MAPK activation was markedly increased. Regarding the upstream of MAPK, Gab1 phosphorylation was also higher with high beta1 integrin expression, while Shc phosphorylation was not altered. In addition, enhanced phosphatidylinositol 3-kinase activation was also observed. These observations suggest that Gab1 and phosphatidylinositol 3-kinase play pivotal roles in the beta1 integrin-mediated regulation of the epidermal stem cell compartment.     

Isolation and sequence analysis of the Sox-1, -2, -3 homologs in Trionyx sinensis and Alligator sinensis having temperature-dependent sex determination

Members of the Sox gene family are characterized by an HMG-box that shows sequence similarity with that of the mouse testis-determining gene Sry. Using degenerate primers PCR, seven and eight HMG-box motifs of Sry-related genes were cloned and sequenced from genomic DNA of Trionyx sinensis (termed TS41-47) and Alligator sinensis (AS41-48) with TSD (temperature-dependent sex determination). Among 15 Sry-related genes, TS41, TS42, AS41, and AS42 shared 80, 72, 81, and 79% amino acid identity, respectively, with each HMG-box domain of the mouse Sox-1, -2, and -3 genes by Blast analysis. Molecular phylogenetic analysis showed that the clustering of TS41-42 and AS41-42 was distant to the clustering of the nonreptilian vertebrate Sox-1, -2, -3 homologs, including fish, amphibian, bird, and mammals. The amino acid identity among TS41-42, AS41-42, and the nonreptilian vertebrate Sox-1, -2, -3 homologs is lower than identities among the Sox-1, -2, -3 homologs, suggesting that the sequence changes in TS41-42 of Trionyx sinensis and AS41-42 of Alligator sinensis might have occurred after the diversification of amniotes.     

Analysis of the L1-deficient mouse phenotype reveals cross-talk between Sema3A and L1 signaling pathways in axonal guidance

In humans, defects of the corticospinal tract have been attributed to mutations in the gene encoding L1 CAM, a phenotype that is reproduced in L1-deficient mice. Using coculture assays, we report that Sema3A secreted from the ventral spinal cord repels cortical axons from wild-type but not from L1-deficient mice. L1 and neuropilin-1 (NP-1) form a stable complex, and their extracellular domains can directly associate. Thus, L1 is a component of the Sema3A receptor complex, and L1 mutations may disrupt Sema3A signaling in the growth cone, leading to guidance errors. Addition of soluble L1Fc chimeric molecules does not restore Sema3A responsiveness of L1-deficient axons; instead, it converts the repulsion of wild-type axons into an attraction, further supporting a function for L1 in the Sema3A transducing pathways within the growth cone.     

Inhibition of neutrophil apoptosis by type 1 IFN depends on cross-talk between phosphoinositol 3-kinase,protein kinase C-delta,and NF-kappa B signaling pathways

Neutrophils are abundant, short-lived leukocytes with a key role in the defense against rapidly dividing bacteria. They enter apoptosis spontaneously within 24-48 h of leaving the bone marrow. However, their life span can be extended during inflammatory responses by several proinflammatory cytokines. Inappropriate survival of neutrophils contributes to chronic inflammation and tissue damage associated with diseases such as rheumatoid arthritis. We have previously reported that type I IFNs can inhibit both cytokine deprivation and Fas-induced apoptosis in activated T cells. IFN-beta locally produced by hyperplastic fibroblasts within the pannus tissue of patients with rheumatoid arthritis contributes to the inappropriately extended life span of infiltrating T cells. Type I IFNs are equally effective at delaying spontaneous apoptosis in human neutrophils. In the work presented here we investigated the signaling pathways involved in mediating this effect. The antiapoptotic actions of IFN-beta were targeted at an early stage of neutrophil apoptosis, occurring upstream of mitochondrial permeability transition, and were phosphatidylinositol 3-kinase (PI3K) dependent, as they were blocked by the PI3K inhibitor LY294002. Analysis of signaling pathways downstream of PI3K revealed that the antiapoptotic effect of type 1 IFN was inhibited by rottlerin, SN50, and cycloheximide, indicating requirements for activation of protein kinase C-delta, NF-kappaB, and de novo protein synthesis, respectively. Moreover, EMSA was used to show that the activation of NF-kappaB occurred downstream of PI3K and protein kinase C-delta activation. We conclude that type I IFNs inhibit neutrophil apoptosis in a PI3K-dependent manner, which requires activation of protein kinase C-delta and induction of NF-kappaB-regulated genes.