A copper-loaded self-assembled nanoparticle for disturbing the tumor redox balance and triple anti-tumor therapy.

Both chemodynamic therapy and photodynamic therapy, based on the production of reactive oxygen (ROS), have excellent potential in cancer therapy. However, the abnormal redox homeostasis in tumor cells, especially the overexpressed glutathione (GSH) could scavenge ROS and reduce the anti-tumor efficiency. Therefore, it is essential to develop a simple and effective tumor-specific drug delivery system for modulating the tumor microenvironment (TME) and achieving synergistic therapy at the tumor site. In this study, self-assembled nanoparticles (named CDZP NPs) were developed using copper ion (Cu2+), doxorubicin (Dox), zinc phthalocyanine (ZnPc) and a trace amount of poly(2-(di-methylamino)ethylmethacrylate)-poly[(R)-3-hydroxybutyrate]-poly(2-(dimethylamino)ethylmethacrylate) (PDMAEMA-PHB-PDMAEMA) through chelation, π-π stacking and hydrophobic interaction. These triple factor-responsive (pH, laser and GSH) nanoparticles demonstrated unique advantages through the synergistic effect. Highly controllable drug release ensured its effectiveness at the tumor site, Dox-induced chemotherapy and ZnPc-mediated fluorescence (FL) imaging exhibited the distribution of nanoparticles. Meanwhile, Cu2+-mediated GSH-consumption not only reduced the intracellular ROS elimination but also produced Cu+ to catalyze hydrogen peroxide (H2O2) and generated hydroxyl radicals (˙OH), thereby enhancing the chemodynamic and photodynamic therapy. Herein, this study provides a green and relatively simple method for preparing multifunctional nanoparticles that can effectively modulate the TME and improve synergetic cancer therapy.

PPR596 Is Required for nad2 Intron Splicing and Complex I Biogenesis in Arabidopsis.

Mitochondria are essential organelles that generate energy via oxidative phosphorylation. Plant mitochondrial genome encodes some of the respiratory complex subunits, and these transcripts require accurate processing, including C-to-U RNA editing and intron splicing. Pentatricopeptide repeats (PPR) proteins are involved in various organellar RNA processing events. PPR596, a P-type PPR protein, was previously identified to function in the C-to-U editing of mitochondrial rps3 transcripts in Arabidopsis. Here, we demonstrate that PPR596 functions in the cis-splicing of nad2 intron 3 in mitochondria. Loss of the PPR596 function affects the editing at rps3eU1344SS, impairs nad2 intron 3 splicing and reduces the mitochondrial complex I's assembly and activity, while inducing alternative oxidase (AOX) gene expression. This defect in nad2 intron splicing provides a plausible explanation for the slow growth of the ppr595 mutants. Although a few P-type PPR proteins are involved in RNA C-to-U editing, our results suggest that the primary function of PPR596 is intron splicing.

PHB3 Is Required for the Assembly and Activity of Mitochondrial ATP Synthase in Arabidopsis.

Mitochondrial ATP synthase is a multiprotein complex, which consists of a matrix-localized F1 domain (F1-ATPase) and an inner membrane-embedded Fo domain (Fo-ATPase). The assembly process of mitochondrial ATP synthase is complex and requires the function of many assembly factors. Although extensive studies on mitochondrial ATP synthase assembly have been conducted on yeast, much less study has been performed on plants. Here, we revealed the function of Arabidopsis prohibitin 3 (PHB3) in mitochondrial ATP synthase assembly by characterizing the phb3 mutant. The blue native PAGE (BN-PAGE) and in-gel activity staining assays showed that the activities of ATP synthase and F1-ATPase were significantly decreased in the phb3 mutant. The absence of PHB3 resulted in the accumulation of the Fo-ATPase and F1-ATPase intermediates, whereas the abundance of the Fo-ATPase subunit a was decreased in the ATP synthase monomer. Furthermore, we showed that PHB3 could interact with the F1-ATPase subunits ß and δ in the yeast two-hybrid system (Y2H) and luciferase complementation imaging (LCI) assay and with Fo-ATPase subunit c in the LCI assay. These results indicate that PHB3 acts as an assembly factor required for the assembly and activity of mitochondrial ATP synthase.

A mutation in the promoter of the arabinogalactan protein 7-like gene PcAGP7-1 affects cell morphogenesis and brassinolide content in pear (Pyrus communis L.) stems.

Dwarfing rootstocks and dwarf cultivars are urgently needed for modern pear cultivation. However, germplasm resources for dwarfing pear are limited, and the underlying mechanisms remain unclear. We previously showed that dwarfism in pear is controlled by the single dominant gene PcDw (Dwarf). We report here that the expression of PcAGP7-1 (ARABINOGALACTAN PROTEIN 7-1), a key candidate gene for PcDw, is significantly higher in dwarf-type pear plants because of a mutation in an E-box in the promoter. Electrophoretic mobility shift assays and transient infiltration showed that the transcription factors PcBZR1 and PcBZR2 could directly bind to the E-box of the PcAGP7-1 promoter and repress transcription. Moreover, transgenic pear lines overexpressing PcAGP7-1 exhibited obvious dwarf phenotypes, whereas RNA interference pear lines for PcAGP7-1 were taller than controls. PcAGP7-1 overexpression also enhanced cell wall thickness, affected cell morphogenesis, and reduced brassinolide (BL) content, which inhibited BR signaling via a negative feedback loop, resulting in further dwarfing. Overall, we identified a dwarfing mechanism in perennial woody plants involving the BL-BZR/BES-AGP-BL regulatory module. Our findings provide insight into the molecular mechanism of plant dwarfism and suggest strategies for the molecular breeding of dwarf pear cultivars.

Em pty Pericarp24 and Empty Pericarp25 Are Required for the Splicing of Mitochondrial Introns, Complex I Assembly, and Seed Development in Maize.

RNA splicing is an essential post-transcriptional regulation in plant mitochondria and chloroplasts. As the mechanism of RNA splicing remains obscure, identification and functional elucidation of new splicing factors are necessary. Through a characterization of two maize mutants, we cloned Empty pericarp 24 (Emp24) and Empty pericarp 25 (Emp25). Both Emp24 and Emp25 encode mitochondrion-targeted P-type PPR proteins. EMP24 is required for the splicing of nad4 introns 1 and 3, which was reported (Ren Z. et al., 2019), and EMP25 functions in the splicing of nad5 introns 1, 2, and 3. Absence of either Nad4 or Nad5 proteins blocks the assembly of mitochondrial complex I, resulting in the formation of a sub-sized complex I of similar size in both mutants. Mass spectrometry identification revealed that the subcomplexes in both mutants lack an identical set of proteins of complex I. These results indicate that EMP24 and EMP25 function in the splicing of nad4 and nad5 introns, respectively, and are essential to maize kernel development. The identification of the subcomplexes provides genetic and molecular insights into the modular complex I assembly pathway in maize.

Composition of Mitochondrial Complex I during the Critical Node of Seed Aging in Oryza sativa.

Previous studies have documented mitochondrial dysfunction during the critical node (CN) of rice (Oryza sativa) seed aging, including a decrease in the capacity of NADH dependent O2 consumption. This raises the hypothesis that changes in the activity of NADH:ubiquinone oxidoreductase (complex I) may play a role in seed aging. The composition and activity of complex I was investigated at the CN of aged rice seeds. Using BN-PAGE and SWATH-MS 52 complex I subunits were identified, nineteen for the first time to be experimentally detected in rice. The subunits of the matrix arm (N and Q modules) were reduced in abundance at the CN, in accordance with a reduction in the capacity to oxidise NADH, reducing substrate oxidation and increase ROS accumulation. In contrast, subunits in the P module increased in abundance that contains many mitochondrial encoded subunits. It is proposed that the changes in complex I abundance subunits may indicate a premature re-activation of mitochondrial biogenesis, as evidenced by the increase in mitochondrial encoded subunits. This premature activation of mitochondrial biogenesis may under-pin the decreased viability of aged seeds, as mitochondrial biogenesis is a crucial event in germination to drive growth before autotrophic growth of the seedling is established.

Comprehensive Mitochondrial Metabolic Shift during the Critical Node of Seed Ageing in Rice.

The critical node (CN) in seed aging in rice (Oryza sativa) is the transformation from Phase I (P-I) to Phase II (P-II) of the reverse S-shaped curve (RS-SC). Although mitochondrial dysfunction plays a key role in seed ageing, the metabolic shift in the CN remains poorly understood. Here, we investigated the mitochondrial regulatory mechanisms during the CN of rice seed ageing. We showed that during the CN of seed ageing, the mitochondrial ultrastructure was impaired, causing oxygen consumption to decrease, along with cytochrome c (cyt c) oxidase and malate dehydrogenase (MDH) activity. In addition, the transcript levels for the alternative pathway of the electron transport chain (ETC) were significantly induced, whereas the transcripts of the cytochrome oxidase (COX) pathway were inhibited. These changes were concomitant with the down-regulation of mitochondrial protein levels related to carbon and nitrogen metabolism, ATP synthase (ATPase) complex, tricarboxylic acid cycle (TCA) cycle, mitochondrial oxidative enzymes, and a variety of other proteins. Therefore, while these responses inhibit the production of ATP and its intermediates, signals from mitochondria (such as the decrease of cyt c and accumulation of reactive oxygen species (ROS)) may also induce oxidative damage. These events provide considerable information about the mitochondrial metabolic shifts involved in the progression of seed ageing in the CN.