In an attempt to improve stress tolerance of tomato (Lycopersicon esculentum) plants, an expression vector containing an Arabidopsis C-repeat/dehydration responsive element binding factor 1 (CBF1) cDNA driven by a cauliflower mosaic virus 35S promoter was transferred into tomato plants. Transgenic expression of CBF1 was proved by northern-and western-blot analyses. The degree of chilling tolerance of transgenic T 1 and T 2 plants was found to be significantly greater than that of wild-type tomato plants as measured by survival rate, chlorophyll fluorescence value, and radical elongation. The transgenic tomato plants exhibited patterns of growth retardation; however, they resumed normal growth after GA 3 (gibberellic acid) treatment. More importantly, GA 3 -treated transgenic plants still exhibited a greater degree of chilling tolerance compared with wild-type plants. Subtractive hybridization was performed to isolate the responsive genes of heterologous Arabidopsis CBF1 in transgenic tomato plants. CATALASE1 (CAT1) was obtained and showed activation in transgenic tomato plants. The CAT1 gene and catalase activity were also highly induced in the transgenic tomato plants. The level of H 2 O 2 in the transgenic plants was lower than that in the wild-type plants under either normal or cold conditions. The transgenic plants also exhibited considerable tolerance against oxidative damage induced by methyl viologen. Results from the current study suggest that heterologous CBF1 expression in transgenic tomato plants may induce several oxidative-stress responsive genes to protect from chilling stress.Many tropical and subtropical crops, e.g. tomato (Lycopersicon esculentum), bell pepper (Capsicum annuum), and avocado (Persea americana), are sensitive to cold (Saltveit and Morris, 1990). Because of the susceptibility to chilling, the growing season of the crops is limited, and the quality of produce is affected. On the other hand, plants originating in the temperate zone are generally more tolerant to cold and have protective processes, such as cold acclimation (Thomashow, 1999). It has been demonstrated that the ability to cold acclimate is related to specific signal transduction pathways resulting in the activation of many cold-regulated (COR) genes (Thomashow, 1998).The COR genes, including RD29A (COR78), COR15a, KIN1, and COR6.6 of Arabidopsis, are inducible in response to cold treatment, ABA, and water-deficit stress (Thomashow, 1998). The C-repeat (CRT) and dehydration responsive element (DRE)-related motifs have been reported in the promoter sequences of these genes (Horvath et al., 1993;Nordin et al., 1993; Baker et al., 1994;Wang et al., 1995). The CRT/DRE binding factor 1 (CBF1) has been isolated using a yeast (Saccharomyces cerevisiae) onehybrid system (Stockinger et al., 1997). Overexpression of CBF1 can induce COR gene expression and result in increased tolerance to freezing temperature treatment without cold-acclimation (Jaglo-Ottosen et al., 1998). Arabidopsis CBF1 is also heterologously effective in canola ...
About half of all melanomas harbor a mutation that results in a constitutively active BRAF kinase mutant (BRAF V600E/K ) that can be selectively inhibited by targeted BRAF inhibitors (BRAFis). While patients treated with BRAFis initially exhibit measurable clinical improvement, the majority of patients eventually develop drug resistance and relapse. Here, we observed marked elevation of WNT5A in a subset of tumors from patients exhibiting disease progression on BRAFi therapy. WNT5A transcript and protein were also elevated in BRAFiresistant melanoma cell lines generated by long-term in vitro treatment with BRAFi. RNAi-mediated reduction of endogenous WNT5A in melanoma decreased cell growth, increased apoptosis in response to BRAFi challenge, and decreased the activity of prosurvival AKT signaling. Conversely, overexpression of WNT5A promoted melanoma growth, tumorigenesis, and activation of AKT signaling. Similarly to WNT5A knockdown, knockdown of the WNT receptors FZD7 and RYK inhibited growth, sensitized melanoma cells to BRAFi, and reduced AKT activation. Together, these findings suggest that chronic BRAF inhibition elevates WNT5A expression, which promotes AKT signaling through FZD7 and RYK, leading to increased growth and therapeutic resistance. Furthermore, increased WNT5A expression in BRAFi-resistant melanomas correlates with a specific transcriptional signature, which identifies potential therapeutic targets to reduce clinical BRAFi resistance.
Cyclic GMP (cGMP) is an important regulator in eukaryotes, and cGMP-dependent protein kinase (PKG) plays a key role in perceiving cellular cGMP in diverse physiological processes in animals. However, the molecular identity, property, and function of PKG in plants remain elusive. In this study, we have identified PKG from plants and characterized its role in mediating the gibberellin (GA) response in rice (Oryza sativa). PKGs from plants are structurally unique with an additional type 2C protein phosphatase domain. Rice PKG possesses both protein kinase and phosphatase activities, and cGMP stimulates its kinase activity but inhibits its phosphatase activity. One of PKG's targets is GAMYB, a transcription factor in GA signaling, and the dual activities of PKG catalyze the reversible phosphorylation of GAMYB at Ser 6 and modulate the nucleocytoplasmic distribution of GAMYB in response to GA. Loss of PKG impeded the nuclear localization of GAMYB and abolished GAMYB function in the GA response, leading to defects in GA-induced seed germination, internode elongation, and pollen viability. In addition to GAMYB, PKG has multiple potential targets and thus has broad effects, particularly in the salt stress response.
Sulfoquinovosyltransferase 2 (SQD2) catalyses the final step in the sulfoquinovosyldiacylglycerol (SQDG) biosynthetic pathway. It is involved in the phosphate starvation response. Here, we show that rice SQD2.1 has dual activities catalysing SQDG synthesis and flavonoid glycosylation. SQD2.1 null mutants (sqd2.1) in rice had decreased levels of glycosidic flavonoids, particularly apigenin 7‐O‐glucoside (A7G), whereas these metabolites were increased in rice plants overexpressing SQD2.1. The sqd2.1 mutants and SQD2.1 overexpressing lines showed reduced and enhanced, respectively, tolerance to salinity and drought. Treating the sqd2.1 mutants with A7G decreased oxidative damage and restored stress tolerance to the wild‐type levels. These findings demonstrate that SQD2.1 has a novel function in the glycosylation of flavonoids that is required for osmotic stress tolerance in rice. The novel activity of SQD2.1 in the production of glycosidic flavonoids improves scavenging of reactive oxygen species and protects against excessive oxidation.
Elevated levels of nuclear β-catenin are associated with higher rates of survival in patients with melanoma, raising questions as to how ß-catenin is regulated in this context. In the present study, we investigated the formal possibility that the secretion of WNT ligands that stabilize ß-catenin may be regulated in melanoma and thus contributes to differences in ß-catenin levels. We find that WLS, a conserved transmembrane protein necessary for WNT secretion, is decreased in both melanoma cell lines and in patient tumours relative to skin and to benign nevi. Unexpectedly, reducing endogenous WLS with shRNAs in human melanoma cell lines promotes spontaneous lung metastasis in xenografts in mice and promotes cell proliferation in vitro. Conversely, overexpression of WLS inhibits cell proliferation in vitro. Activating β-catenin downstream of WNT secretion blocks the increased cell migration and proliferation observed in the presence of WLS shRNAs, while inhibiting WNT signalling rescues the growth defects induced by excess WLS. These data suggest that WLS functions as a negative regulator of melanoma proliferation and spontaneous metastasis by activating WNT/β-catenin signalling.
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