Abstract The changing climate leads to frequent low temperature events, which results in lower crop production worldwide. Plant growth and development are regulated by WRKYs, as well as a variety of defense responses, including salt, drought, and extreme temperature stresses. Here, a transcription factor AeWRKY31 was obtained from Abelmoschus esculentus and ectopic expressed in Arabidopsis thaliana . Molecular localization and self-transactivation assays revealed AeWRKY31 was a nuclear-localized protein with self-transactivation property. Subsequently, AeWRKY31 expression activated the expression of AtABA1 , AtABA2, and AtNCED6 , and promoted endogenous ABA accumulation, which induced stomata closure of transgenic Arabidopsis. Histochemical staining and ROS content determination demonstrated that AeWRKY31 expression inhibited the accumulation of H 2 O 2 and O 2- and decrease electrolyte leakage in transgenic lines. Meanwhile, compared with that of WT, relative expression levels of cold response-related genes were up-regulated in transgenic lines. Finally, AeWRKY31 transgenic Arabidopsis plants exhibited enhanced cold tolerance both in vivo and in vitro experiments. Our findings will benefit molecular breeding and anti-cold mechanism investigation of okra.
As an indispensable inflammatory mediator during sepsis, granulocyte colony-stimulating factor (G-CSF) facilitates neutrophil production by activating G-CSFR. However, little is known about the role of intracellular downstream signalling pathways in the induction of inflammation. To explore the functions of molecules in regulating G-CSFR signalling, RNA sequencing and integrated proteomic and phosphoproteomic analyses were conducted to predict the differentially expressed molecules in modulating the inflammatory response after G-CSFR expression was either up- or downregulated, in addition to the confirmation of their biological function by diverse experimental methods. In the integrated bioinformatic analysis, 3190 differentially expressed genes (DEGs) and 1559 differentially expressed proteins (DEPs) were identified in multiple-group comparisons (p < 0.05, FC > ± 1.5) using enrichment analyses, as well as those classic pathways such as the TNF, NFkappaB, IL-17, and TLR signalling pathways. Among them, 201 proteins, especillay intercellular cell adhesion molecule-1 (ICAM1) and PKCa, were identified as potential molecules involved in inflammation according to the protein–protein interaction (PPI) analysis, and the leukocyte transendothelial migration (TEM) pathway was attributed to the intervention of G-CSFR. Compared with the control and TNF-a treatment, the G-CSFR (G-CSFROE)-overexpressing led to an obvious increase in the number of leukocytes with the TEM phenotype. Mechanically, the expression of ICAM1 and PKCa was significantly up- and downregulated by G-CSFROE, which directly led to increased TEM; moreover, PKCa expression was negatively regulated by ICAM1 expression, leading to aberrant leukocyte TEM. Altogether, the ICAM1‒PKCa axis was found a meaningful target in the leukocyte TEM induced by G-CSFR upregulation.
Cynanchum auriculatum, an early food-medicine homologous plant native to Asia, possesses significant nutritional and health benefits. However, the presence of cadmium (Cd) in the soil poses a hazard to the germination and growth of C. auriculatum. As nitric oxide (NO) plays a vital role in plant resistance to heavy metal stress, we used three different concentrations of SNP treatment during the germination phase, aiming to alleviate the inhibitory effects of Cd stress on the seed germination of C. auriculatum. The results indicated that when compared to seeds treated with SNP concentrations of 0.2 mM and 0.8 mM, C. auriculatum seeds treated with 0.4 mM SNP exhibited an improved germination rate and germination index, as well as longer hypocotyl. Furthermore, in comparison to NOS-like, the SNP application stimulated the production of endogenous NO through NR catalysis. Additional investigations showed that the ABA level decreased while the GA level increased under normal conditions, while the SNP application enhanced the accumulation of both ABA and GA in C. auriculatum seeds under Cd stress. Histochemical staining and biochemical indicators demonstrated that SNP treatment enhanced the enzymatic activity of SOD, POD, and CAT, while inhibiting the production of hydrogen peroxide and superoxide anion. Moreover, SNP treatment resulted in increased α-amylase activity, which facilitated starch hydrolysis and the generation of soluble sugar. Ultimately, the seed vitality of C. auriculatum under Cd stress was promoted. Our findings present a theoretical framework for the application of SNP in the seed germination mechanism of C. auriculatum and establish the groundwork for comprehending the physiological role of NO under Cd stress.
Okra (Abelmoschus esculentus L.) is a particular vegetable with both edible and medicinal values. However, the expression pattern of the okra reference genes in response to abiotic stress has not been explored. In the present study, 18 potential reference genes were selected from okra in various tissues and abiotic stress conditions, and their expression levels were detected by Real-Time quantitative PCR (RT-qPCR). Their expression stabilities were calculated by four algorithms (geNorm, NormFinder, BestKeeper, and RefFinder). Under cold stress, the most stable genes included GAPC1 and CYP (leaf), CYP and ACT7 (root), HIS6 and GAPC1 (stem), and HIS6 and 60s (different tissues). Under salt stress, EF-1α and UBQ (leaf), EF-1α and UBQ (root), TUA4 and Eif (stem), and HIS6 and Eif (different tissues) were the most stable genes. Under drought stress, UBQ and Eif in the leaf, HIS6 and Eif in the root, TUA4 and HIS6 in the stem, and UBQ and Eif in different tissues were most stably expressed in okra. In addition, complete sequencing results by RefFinder showed that HIS6 and ACT7 in the leaf, HIS6 and Eif in the root, UBC5B and 60s in the stem, and HIS6 and Eif in different tissues, were most the suitable reference genes for okra. Furthermore, AeMYB1R1 transcription factor was used to verify the reliability of RT-qPCR values. In summary, this study was carried out to demonstrate the potential reference genes of okra under abiotic stress, aiming to provide a molecular basis for functional gene analysis and regulatory mechanism research of okra.
Osteoarthritis (OA) is a debilitating joint disorder characterized by progressive cartilage degeneration. During OA, subchondral bone undergoes microstructural and molecular changes that precede cartilage degradation. However, specific mechanisms underlying metabolic dysregulation of the bone-cartilage unit remain unclear. This study aims to investigate the role of receptor-interacting protein kinase-3 (RIP3) in OA progression, focusing on bone-cartilage metabolic homeostasis. RIP3-mediated pathological and metabolic alterations in chondrocytes, osteoblasts, and bone marrow-derived macrophages (BMMs) were evaluated. RIP3-mediated OA manifestations in cartilage and more importantly, subchondral bone were determined by intra-articular overexpression of RIP3 in rats. The protective effect of RIP3 deficiency on the bone-cartilage unit during OA was systematically investigated using Rip3 knockout mice. The CMap database was used to screen for compounds that abrogate RIP3-induced OA pathological changes. RIP3 was upregulated in the cartilage and subchondral bone of OA patients and post-traumatic OA mouse model. RIP3 overexpression not only inhibited extracellular matrix (ECM) anabolism in chondrocytes but also attenuated osteoblast differentiation, whereas RIP3 deficiency blunted receptor activator of NF-kappaB ligand-mediated osteoclastogenesis of BMMs. Intra-articular RIP3 overexpression induced the imbalance of SP7+ osteoblasts/tartrate-resistant acid phosphatase (TRAP)+ osteoclasts within the subchondral bone in addition to cartilage degeneration in rats, while Rip3 deletion significantly improved structural outcomes of the bone-cartilage unit, and achieved pain relief as well as functional improvement in surgery-induced and spontaneous OA mouse models. Mechanistically, RIP3 initiates OA by perturbing critical events, including cartilage metabolism, inflammatory responses, senescence, and osteoclast differentiation. Clofibrate, a hypolipidemic drug, was identified as a novel RIP3 inhibitor that reverses ECM catabolism in OA. RIP3 is an essential governor of whole joint metabolic homeostasis by regulating both cartilage metabolism and subchondral bone remodeling. Reconstruction of the bone-cartilage unit by targeting RIP3 might provide a two-birds-one-stone approach for the development of future OA therapies.
Abstract Background Water shortage caused by global warming seriously affects the yield and quality of vegetable crop. β-carotene, the lipid-soluble natural product with important pharmacological value, is abundant in celery. Transcription factor MYB family extensively disperses in plants and plays regulatory roles in carotenoid metabolism and water scarcity response. Results Here, AgMYB5 gene encoding 196 amino acids was amplified from celery cv. ‘Jinnanshiqin’. In celery, the expression of AgMYB5 exhibited transactivation activity, tissue specificity, and drought-condition responsiveness. Further analysis proved that ectopic expression of the AgMYB5 increased β-carotene content and promoted drought resistance in transgenic Arabidopsis thaliana . Moreover, AgMYB5 expression promoted β-carotene biosynthesis by triggering the expression of AtCRTISO and AtLCYB , which in turn increased antioxidant enzyme activities, and led to the decreased contents of H 2 O 2 and MDA, and the inhibition of O 2− generation. Meanwhile, β-carotene accumulation promoted endogenous ABA biosynthesis of the transgenic Arabidopsis plants overexpression AgMYB5 gene, which resulted in ABA-induced stomatal closing and delayed water loss rate. In addition, ectopic expression of AgMYB5 increased expression levels of AtERD1 , AtP5CS1 , AtRD22 , and AtRD29. Conclusions The findings indicated that AgMYB5 up-regulated β-carotene biosynthesis and drought resistance of Arabidopsis .
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