The class II KNOX transcription factors KNAT3 and KNAT7 synergistically regulate monolignol biosynthesis in Arabidopsis.

The function of the transcription factor KNOTTED ARABIDOPSIS THALIANA7 (KNAT7) is still unclear since it appears to be either a negative or a positive regulator for secondary cell wall deposition with its loss-of-function mutant displaying thicker interfascicular and xylary fiber cell walls but thinner vessel cell walls in inflorescence stems. To explore the exact function of KNAT7, class II KNOTTED1-LIKE HOMEOBOX (KNOX II) genes in Arabidopsis including KNAT3, KNAT4, and KNAT5 were studied together. By chimeric repressor technology, we found that both KNAT3 and KNAT7 repressors exhibited a similar dwarf phenotype. Both KNAT3 and KNAT7 genes were expressed in the inflorescence stems and the knat3 knat7 double mutant exhibited a dwarf phenotype similar to the repressor lines. A stem cross-section of knat3 knat7 displayed an enhanced irregular xylem phenotype as compared with the single mutants, and its cell wall thickness in xylem vessels and interfascicular fibers was significantly reduced. Analysis of cell wall chemical composition revealed that syringyl lignin was significantly decreased while guaiacyl lignin was increased in the knat3 knat7 double mutant. Coincidently, the knat3 knat7 transcriptome showed that most lignin pathway genes were activated, whereas the syringyl lignin-related gene Ferulate 5-Hydroxylase (F5H) was down-regulated. Protein interaction analysis revealed that KNAT3 and KNAT7 can form a heterodimer, and KNAT3, but not KNAT7, can interact with the key secondary cell wall formation transcription factors NST1/2, which suggests that the KNAT3-NST1/2 heterodimer complex regulates F5H to promote syringyl lignin synthesis. These results indicate that KNAT3 and KNAT7 synergistically work together to promote secondary cell wall biosynthesis.

CRISPR/Cas9 mutated p-coumaroyl shikimate 3'-hydroxylase 3 gene in Populus tomentosa reveals lignin functioning on supporting tree upright.

The lignin plays one of the most important roles in plant secondary metabolism. However, it is still unclear how lignin can contribute to the impressive height of wood growth. In this study, C3'H, a rate-limiting enzyme of the lignin pathway, was used as the target gene. C3'H3 was knocked out by CRISPR/Cas9 in Populus tomentosa. Compared with wild-type popular trees, c3'h3 mutants exhibited dwarf phenotypes, collapsed xylem vessels, weakened phloem thickening, decreased hydraulic conductivity and photosynthetic efficiency, and reduced auxin content, except for reduced total lignin content and significantly increased H-subunit lignin. In the c3'h3 mutant, the flavonoid biosynthesis genes CHS, CHI, F3H, DFR, ANR, and LAR were upregulated, and flavonoid metabolite accumulations were detected, indicating that decreasing the lignin biosynthesis pathway enhanced flavonoid metabolic flux. Furthermore, flavonoid metabolites, such as naringenin and hesperetin, were largely increased, while higher hesperetin content suppressed plant cell division. Thus, studying the c3'h3 mutant allows us to deduce that lignin deficiency suppresses tree growth and leads to the dwarf phenotype due to collapsed xylem and thickened phloem, limiting material exchanges and transport.

10-Hydroxycamptothecin (HCPT) nanosuspensions stabilized by mPEG1000-HCPT conjugate: high stabilizing efficiency and improved antitumor efficacy.

In this study, polyethylene glycol (PEG)ylated 10-hydroxycamptothecin (mPEG1000-HCPT) was synthesized and used as a stabilizer to prepare 10-hydroxycamptothecin (HCPT) nanosuspensions for their in vitro and in vivo antitumor investigation. The resultant HCPT nanosuspensions (HCPT-NSps) had a very high drug payload of 94.90% (w/w) and a mean particle size of 92.90±0.20 nm with narrow size distribution (polydispersity index of 0.16±0.01). HCPT-NSps could be lyophilized without the need of the addition of any cryoprotectant and then be reconstituted into nanosuspensions of a similar size by direct resuspension in water. HCPT was in crystalline form in HCPT-NSps. Using mPEG1000-HCPT as stabilizer, insoluble camptothecin and 7-ethyl-10-hydroxycamptothecin could also be easily made into nanosuspensions with similar features such as high drug payload, small particle size, and cryoprotectant-free freeze drying. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide assay indicated that the HCPT-NSps had a significantly higher cytotoxicity than HCPT injections, with 3.77 times lower IC50 value against HepG2 cells and 14.1 times lower IC50 value against MCF-7 cells. An in vivo study in H22 tumor-bearing mice after intravenous injection of HCPT-NSps demonstrated that HCPT-NSps significantly improved the antitumor efficacy compared to the commercially available HCPT injections (86.38% vs 34.97%) at the same dose of 5 mg/kg. Even at 1/4 of the dose, HCPT-NSps could also achieve a similar antitumor efficacy to that of HCPT injections. mPEG1000-HCPT may be a highly efficient stabilizer able to provide camptothecin-based drugs, and probably other antitumor agents containing aromatic structure, with unique nanosuspensions or nanocrystals for improved in vivo therapeutic efficacy.

High Drug Payload 10-Hydroxycamptothecin Nanosuspensions Stabilized by Cholesterol-PEG: In Vitro and In Vivo Investigation.

The objective of this study was to evaluate the feasibility of producing 10-hydroxycamptothecin nanosuspensions with high drug payload and to then determine the in vitro and in vivo characteristics of these nanosuspensions. Using cholesterol-PEG600 as a stabilizer, 10-hydroxycamptothecin nanosuspensions were successfully prepared using a precipitation-combined high-pressure homogenization method. A satisfactory drug payload of 90.39% (w/w) was achieved. The obtained nanosuspensions were spherical, with a mean particle size of 115.0 ± 0.4 nm, and they were monodisperse with a polydispersity index of 0.134 ± 0.001. The 10-hydroxycamptothecin remained in the same crystalline form in both the nanosuspensions and the bulk powder. In vitro, the 10-hydroxycamptothecin nanosuspensions released the encapsulated drug with nearly zero-order kinetics, and the accumulative release reached 90% within 72 hours. In vitro cytotoxicity assay showed that the 1 0-hydroxycamptothecin nanosuspensions had significantly enhanced cytotoxicity against HepG2 cells compared to the commercially available 10-hydroxycamptothecin injections. The in vivo study with H22 tumor-bearing mice and intravenous injection of the drug showed that in contrast to the 10-hydroxycamptothecin injections, the 10-hydroxycamptothecin nanosuspensions exhibited significantly enhanced biodistribution, particularly in the lung (393.40-fold AUC(0-24h)), liver (192.35-fold AUC(0-24h)), spleen (141.67-fold AUC(0-24h)) and tumor (64.21-fold AUC(0-24h)). The 10-hydroxycamptothecin nanosuspensions also showed improved antitumor therapeutic efficacy over the injections (89.83% vs. 30.56%). This suggests that cholesterol-PEG600 may be an effective stabilizer for the preparation of hydrophobic drug nanosuspensions and that 10-hydroxycamptothecin nanosuspensions are a promising drug delivery system for tumor treatment.