Characterization, evolution, and abiotic stress responses of leucine-rich repeat receptor-like protein kinases (LRR-RLK) in Liriodendron chinense.

BACKGROUND: Liriodendron chinense is susceptible to extinction due to the increasing severity of abiotic stresses resulting from global climate change, consequently impacting its growth, development, and geographic distribution. However, the L. chinense remains pivotal in both socio-economic and ecological realms. The LRR-RLK (leucine-rich repeat receptor-like protein kinase) genes, constituting a substantial cluster of receptor-like kinases in plants, are crucial for plant growth and stress regulation and are unexplored in the L. chinense. RESULT: 233 LchiLRR-RLK genes were discovered, unevenly distributed across 17 chromosomes and 24 contigs. Among these, 67 pairs of paralogous genes demonstrated gene linkages, facilitating the expansion of the LchiLRR-RLK gene family through tandem (35.82%) and segmental (64.18%) duplications. The synonymous and nonsynonymous ratios showed that the LchiLRR-RLK genes underwent a purifying or stabilizing selection during evolution. Investigations in the conserved domain and protein structures revealed that the LchiLRR-RLKs are highly conserved, carrying conserved protein kinase and leucine-rich repeat-like domians that promote clustering in different groups implicating gene evolutionary conservation. A deeper analysis of LchiLRR-RLK full protein sequences phylogeny showed 13 groups with a common ancestor protein. Interspecies gene collinearity showed more orthologous gene pairs between L. chinense and P. trichocarpa, suggesting various similar biological functions between the two plant species. Analysis of the functional roles of the LchiLRR-RLK genes using the qPCR demonstrated that they are involved in cold, heat, and salt stress regulation, especially, members of subgroups VIII, III, and Xa. CONCLUSION: Conclusively, the LRR-RLK genes are conserved in L. chinense and function to regulate the temperature and salt stresses, and this research provides new insights into understanding LchiLRR-RLK genes and their regulatory effects in abiotic stresses.

Physiological and molecular response and tolerance of Macleaya cordata to lead toxicity.

BACKGROUND: Macleaya cordata is a traditional medicinal herb, and it has high tolerance and accumulation ability to heavy metals, which make it a good candidate species for studying phytoremediation. The objectives of this study were to investigate response and tolerance of M. cordata to lead (Pb) toxicity based on comparative analysis of transcriptome and proteome. RESULTS: In this study, the seedlings of M. cordata cultured in Hoagland solution were treated with 100 µmol·L- 1 Pb for 1 day (Pb 1d) or 7 days (Pb 7d), subsequently leaves of M. cordata were taken for the determination of Pb accumulation and hydrogen peroxide production (H2O2), meanwhile a total number of 223 significantly differentially expressed genes (DEGs) and 296 differentially expressed proteins (DEPs) were screened between control and Pb treatments. The results showed leaves of M. cordata had a special mechanism to maintain Pb at an appropriate level. Firstly, some DEGs were iron (Fe) deficiency-induced transporters, for example, genes of vacuolar iron transporter and three ABC transporter I family numbers were upregulated by Pb, which can maintain Fe homeostasis in cytoplasm or chloroplast. In addition, five genes of calcium (Ca2+) binding proteins were downregulated in Pb 1d, which may regulate cytoplasmic Ca2+ concentration and H2O2 signaling pathway. On the other hand, the cysteine synthase upregulated, glutathione S-transferase downregulated and glutathione reductase downregulated in Pb 7d can cause reduced glutathione accumulation and decrease Pb detoxification in leaves. Furthermore, DEPs of eight chlorophyll a/b binding proteins, five ATPases and eight ribosomal proteins can play a pivotal role on chloroplast turnover and ATP metabolism. CONCLUSIONS: Our results suggest that the proteins involved in Fe homeostasis and chloroplast turnover in mesophyll cells may play key roles in tolerance of M. cordata to Pb. This study offers some novel insights into Pb tolerance mechanism of plants, and the potential valuable for environmental remediation of this important medicinal plant.

Non-targeted metabolomics analysis of fermented traditional Chinese medicine and its impact on growth performance, serum biochemistry, and intestinal microbiome of weaned lambs.

Fermented traditional Chinese medicines (TCMs) have been identified as a low-cost and promising feed additive to to alleviate weaning stress in young livestock and poultry effectively. This study investigated the impact of probiotic fermentation on the metabolite content of BanQi (Radix Isatidis and Astragalus membranaceus) extract while also examined the effects of both fermented-BanQi (FBQ) and unfermented-BanQi (UBQ) on growth performance, serum biochemistry, intestinal villi, and gut microbiota in weaned lambs. This study demonstrated that compared with UBQ, FBQ contained significantly higher levels of free amino acids (e.g., phenylalanine and isoleucine), short peptides (e.g., Val-Leu-Pro-Val-Pro-Gln and Gly-Leu), and the active ingredients (e.g., vindesine and reserpine) (P < 0.05). The addition of FBQ to the diet significantly increased the final body weight and average daily gain of weaned lambs (P < 0.05). In addition, FBQ significantly increased the total protein level in the serum and the villus length of the jejunum and ileum in lambs, while significantly reduced the levels of aspartate aminotransferase (AST) and urea (P < 0.05). Sequencing of the intestinal flora showed that FBQ improved the diversity of intestinal flora and promoted the enrichment of beneficial bacteria in the lamb intestine, such as Mogibacterium and Butyrivibrio, compared to NC or UBQ groups (P < 0.05). Fermentation with Bacillus subtilis can enhance the content of free amino acids, peptides, and active ingredients in BanQi extract, making it an effective method to improve the efficacy of traditional Chinese medicine. Adding FBQ to the diet can improve the growth performance of weaned lambs, and its mechanism may be related to increasing the height of intestinal villi and increasing the diversity of intestinal flora.

Design, synthesis and biological evaluation of 4-(indolin-1-yl)-6-substituted-pyrido[3,2-d]pyrimidine derivatives as Mnk1/2 inhibitors.

The Mnk-eIF4E axis plays a crucial role in tumor development, and inhibiting Mnk kinases is a promising approach for cancer therapy. Starting with fragment WS23, a series of 4-(indolin-1-yl)-6-substituted-pyrido[3,2-d]pyrimidine derivatives were designed and synthesized. Among these derivatives, compound 15b showed the highest potency with IC50 values of 0.8 and 1.5 nM against Mnk1 and Mnk2, respectively. Additionally, it demonstrated good selectivity among 30 selected kinases. 15b significantly suppressed MOLM-13 and K562 cell lines growth and caused cell cycle arrest. Furthermore, the Western blot assay revealed that 15b effectively downregulated the downstream proteins p-eIF4E, Mcl-1, and c-myc. Additionally, 15b exhibited remarkable stability in rat plasma and rat and human microsomes. In vivo anti-tumor activity study suggested that treatment with 15b suppressed tumor growth in LL/2 syngeneic models. These findings highlight the potential of 15b as a novel and potent Mnks inhibitor, which deserves further investigation.

Response and Tolerance of Macleaya cordata to Excess Zinc Based on Transcriptome and Proteome Patterns.

Macleaya cordata is a dominant plant of mine tailings and a zinc (Zn) accumulator with high Zn tolerance. In this study, M. cordata seedlings cultured in Hoagland solution were treated with 200 µmol·L-1 of Zn for 1 day or 7 days, and then, their leaves were taken for a comparative analysis of the transcriptomes and proteomes between the leaves of the control and Zn treatments. Differentially expressed genes included those that were iron (Fe)-deficiency-induced, such as vacuolar iron transporter VIT, ABC transporter ABCI17 and ferric reduction oxidase FRO. Those genes were significantly upregulated by Zn and could be responsible for Zn transport in the leaves of M. cordata. Differentially expressed proteins, such as chlorophyll a/b-binding proteins, ATP-dependent protease, and vacuolar-type ATPase located on the tonoplast, were significantly upregulated by Zn and, thus, could be important in chlorophyll biosynthesis and cytoplasm pH stabilization. Moreover, the changes in Zn accumulation, the production of hydrogen peroxide, and the numbers of mesophyll cells in the leaves of M. cordata were consistent with the expression of the genes and proteins. Thus, the proteins involved in the homeostasis of Zn and Fe are hypothesized to be the keys to the tolerance and accumulation of Zn in M. cordata. Such mechanisms in M. cordata can suggest novel approaches to genetically engineering and biofortifying crops.

Precise exogenous insertion and sequence replacements in poplar by simultaneous HDR overexpression and NHEJ suppression using CRISPR-Cas9.

CRISPR-mediated genome editing has become a powerful tool for the genetic modification of biological traits. However, developing an efficient, site-specific, gene knock-in system based on homology-directed DNA repair (HDR) remains a significant challenge in plants, especially in woody species like poplar. Here, we show that simultaneous inhibition of non-homologous end joining (NHEJ) recombination cofactor XRCC4 and overexpression of HDR enhancer factors CtIP and MRE11 can improve HDR efficiency for gene knock-in. Using this approach, the BleoR gene was integrated onto the 3' end of the MKK2 MAP kinase gene to generate a BleoR-MKK2 fusion protein. Based on fully edited nucleotides evaluated by TaqMan real-time PCR, the HDR-mediated knock-in efficiency was up to 48% when using XRCC4 silencing incorporated with a combination of CtIP and MRE11 overexpression compared with no HDR enhancement or NHEJ silencing. Furthermore, this combination of HDR enhancer overexpression and NHEJ repression also increased genome targeting efficiency and gave 7-fold fewer CRISPR-induced insertions and deletions (InDels), resulting in no functional effects on MKK2-based salt stress responses in poplar. Therefore, this approach may be useful not only in poplar and plants or crops but also in mammals for improving CRISPR-mediated gene knock-in efficiency.