RESUMEN
Sugar beet is a significant sugar crop in China, primarily cultivated in arid regions of the north. However, drought often affects sugar beet cultivation, leading to reduced yield and quality. Therefore, understanding the impact of drought on sugar beets and studying their drought tolerance is crucial. Previous research has examined the role of SPL (SQUAMOSA promoter-binding protein-like) transcription factors in plant stress response; however, the precise contribution of SPLs to the drought stress response in sugar beets has yet to be elucidated. In this study, we identified and examined the BvSPL6, BvSPL7, and BvSPL9 genes in sugar beets, investigating their performance during the seedling stage under drought stress. We explored their drought resistance characteristics using bioinformatics, quantitative analysis, physiological experiments, and molecular biology experiments. Drought stress and rehydration treatments were applied to sugar beet seedlings, and the expression levels of BvSPL6, BvSPL7, and BvSPL9 genes in leaves were quantitatively analyzed at 11 different time points to evaluate sugar beets' response and tolerance to drought stress. Results indicated that the expression level of the BvSPL6/9 genes in leaves was upregulated during the mid-stage of drought stress and downregulated during the early and late stages. Additionally, the expression level of the BvSPL7 gene gradually increased with the duration of drought stress. Through analyzing changes in physiological indicators during different time periods of drought stress and rehydration treatment, we speculated that the regulation of BvSPL6/7/9 genes is associated with sugar beet drought resistance and their participation in drought stress response. Furthermore, we cloned the CDS sequences of BvSPL6, BvSPL7, and BvSPL9 genes from sugar beets and conducted sequence alignment with the database to validate the results. Subsequently, we constructed overexpression vectors, named 35S::BvSPL6, 35S::BvSPL7, and 35S::BvSPL9, and introduced them into sugar beets using Agrobacterium-mediated methods. Real-time fluorescence quantitative analysis revealed that the expression levels of BvSPL6/7/9 genes in transgenic sugar beets increased by 40% to 80%. The drought resistance of transgenic sugar beets was significantly enhanced compared with the control group.
Asunto(s)
Beta vulgaris , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas , Plantones , Estrés Fisiológico , Beta vulgaris/genética , Beta vulgaris/fisiología , Sequías , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantones/genética , Estrés Fisiológico/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Microvascular injury resulting from activation and exocytosis are early signs of tissue damage caused by allografting. However, humoral anti-graft reactions are not easily detectable in transplant biopsies. The aim of this study was to establish a bioassay to recapitulate this process in a prospective approach. METHODS: The study was executed by using our previously established protocol to isolate and freeze the donors' microvascular endothelial cells (MVEC) at the transplantation (34 living-related donors and 26 cadaver donors); and to collect sera from the recipients before the transplantation, one-, three- and six-months after transplantation. The activation and exocytosis of the MVEC were determined by incubating the donors' cultures with the recipients' sera. We determined if there was any endothelial activation by quantifying the releases of monocyte chemotactic protein-1 (MCP-1) and interleukin 8 (IL-8) in supernatants and the expressions of membrane intercellular adhesion molecule-1 (CD54) and intercellular adhesion molecule-1 (CD106) by flow cytometry. Endothelial exocytosis was determined by quantifying soluble E-selectin (CD62E) and cytoplasmic von Willebrand Factor (vWF) in supernatants. Endothelial activation or exocytosis was considered positive when the fold change (â§1.5) of post-transplantation to pre-transplantation was reached. We also monitored serum PRA and cytokines using Luminex multiple-plex and cytometric bead-based assay respectively. RESULTS: We found 41.2% recipients (14 out of 34, ranging from 1.5 to 5.2 folds, p < 0.05) exhibited positive MVEC activation in the first month after transplantation as determined by IL-8 levels; 26.5% recipients (9 out of 34, ranging from 1.5 to 11.8 folds, p < 0.05) by MCP-1 levels. In the group of three months post-transplantation, 70.6% patients were positive (12 out of 17, ranging from 1.8 to 87.1 folds, p < 0.05) by IL-8 increased levels; 24% recipients (4 out of 17, ranging from 1.8 to 50.5 folds, p < 0.05) measured by MCP-1 levels. However, these changes disappeared six months after transplantation. Flow cytometric data showed that a time-dependent of CD54+ and CD106+ expressions existed over the course of six months. Most CD54+ and CD106+ cells were CD31- (platelet-endothelial cell adhesion molecule-1), though CD31+/CD106+ (37.5%, 3 out of 8) and CD31+/CD106+ (25%. 2 out of 8) were seen. When comparing donor MVEC activation to their recipient's proinflammatory cytokine levels or PRA status, we could not draw a conclusion regarding the connections between them. The sera collected from recipients at either one- or three-months after allografting did not significantly induce the release of either soluble CD62E or vWF (p > 0.05), indicating exocytosis was not significantly involved in the acute phase of allografting. CONCLUSIONS: This bioassay enables us to detect the activation and exocytosis of donor MVEC elicited by respective sera from mismatched kidney recipients.