Abstract Dynamin‐related proteins (DRPs) are key components of the organelle division machineries, functioning as molecular scissors during the fission process. In Arabidopsis , DRP3A and DRP3B are shared by peroxisomal and mitochondrial division, whereas the structurally‐distinct DRP5B (ARC5) protein is involved in the division of chloroplasts and peroxisomes. Here, we further investigated the roles of DRP3A, DRP3B, and DRP5B in organelle division and plant development. Despite DRP5B's lack of stable association with mitochondria, drp5B mutants show defects in mitochondrial division. The drp3A‐2 drp3B‐2 drp5B‐2 triple mutant exhibits enhanced mitochondrial division phenotypes over drp3A‐2 drp3B‐2 , but its peroxisomal morphology and plant growth phenotypes resemble those of the double mutant. We further demonstrated that DRP3A and DRP3B form a supercomplex in vivo , in which DRP3A is the major component, yet DRP5B is not a constituent of this complex. We thus conclude that DRP5B participates in the division of three types of organelles in Arabidopsis , acting independently of the DRP3 complex. Our findings will help elucidate the precise composition of the DRP3 complex at organelle division sites, and will be instrumental to studies aimed at understanding how the same protein mediates the morphogenesis of distinct organelles that are linked by metabolism.
In this study, we reveal a mechanism by which plants regulate inorganic phosphate (Pi) homeostasis to adapt to environmental changes in Pi availability. This mechanism involves the suppression of a ubiquitin-conjugating E2 enzyme by a specific microRNA, miR399. Upon Pi starvation, the miR399 is upregulated and its target gene, a ubiquitin-conjugating E2 enzyme, is downregulated in Arabidopsis thaliana. Accumulation of the E2 transcript is suppressed in transgenic Arabidopsis overexpressing miR399. Transgenic plants accumulated five to six times the normal Pi level in shoots and displayed Pi toxicity symptoms that were phenocopied by a loss-of-function E2 mutant. Pi toxicity was caused by increased Pi uptake and by translocation of Pi from roots to shoots and retention of Pi in the shoots. Moreover, unlike wild-type plants, in which Pi in old leaves was readily retranslocated to other developing young tissues, remobilization of Pi in miR399-overexpressing plants was impaired. These results provide evidence that miRNA controls Pi homeostasis by regulating the expression of a component of the proteolysis machinery in plants.
There is serious concern about arsenic in the natural environment, which exhibits neurotoxicity and increases the risk of neurodevelopmental disorders. Adverse effects of arsenic have been demonstrated in neurons, but it is not fully understood how arsenic affects other cell types in the brain. In the current study, we examined whether sodium arsenite (NaAsO2) affects the cell cycle, viability, and apoptosis of in vitro-cultured astrocytes isolated from the cerebral cortex of mice. Cultured astrocytes from transgenic mice expressing fluorescent ubiquitination-based cell cycle indicator (Fucci) were subjected to live imaging analysis to assess the effects of NaAsO2 (0, 1, 2, and 4 μM) on the cell cycle and number of cells. Fucci was designed to express monomeric Kusabira Orange2 (mKO2) fused with the ubiquitylation domain of hCdt1, a marker of G1 phase, and monomeric Azami Green (mAG) fused with the ubiquitylation domain of hGem, a marker of S, G2, and M phases. NaAsO2 concentration-dependently decreased the peak levels of the mAG/mKO2 emission ratio when the ratio had reached a peak in astrocytes without NaAsO2 exposure, which was due to attenuating the increase in the mAG-expressing cell number. In contrast, the mAG/mKO2 emission ratio and number of mAG-expressing cells were concentration-dependently increased by NaAsO2 before their peak levels, indicating unscheduled S phase entry. We further examined the fate of cells forced to enter S phase by NaAsO2. We found that most of these cells died up to the end of live imaging. In addition, quantification of the copy number of the glial fibrillary acidic protein gene expressed specifically in astrocytes revealed a concentration-dependent decrease caused by NaAsO2. However, NaAsO2 did not increase the amount of nucleosomes generated from DNA fragmentation and failed to alter the gene expression of molecules relevant to unscheduled S phase entry-coupled apoptosis (p21, p53, E2F1, E2F4, and Gm36566). These findings suggest that NaAsO2 adversely affects the cell cycle and viability of astrocytes by inducing unscheduled S phase entry coupled with cell death that may be caused by mechanisms other than apoptosis.
Abstract The devastating soybean rust (SBR) pathogen, Phakopsora pachyrhizi , encodes many secreted proteins, but only two have been functionally characterized for their roles in rust virulence. Here, we demonstrate that transient expression of P. pachyrhizi effector candidate 15 ( Pp EC15), an aspartic protease, leads to enhanced bacterial growth in planta , suppression of callose deposition, reduced expression of plant defense-related marker genes and suppression of pathogen-associated molecular pattern (PAMP)-induced reactive oxygen species (ROS). Stable expression of Pp EC15 in soybean suppresses PAMP-induced ROS production and enhances bacterial growth, indicating that, collectively, Pp EC15 suppresses host and non-host innate immune responses. Yeast-two-hybrid and proximity labeling identified putative Pp EC15 interacting partners including a peptide-chain release factor (PCRF), a NAC83 (NAM, ATAF, and CUC) transcription factor, and a DAHP (3-deoxy-7-phosphoheptulonate) synthase. We further show that Pp EC15 can cleave DAHP but does not cleave PCRF or NAC83. Virus-induced gene silencing of NAC83, PCRF and DAHP altered PAMP-induced ROS production and salicylic acid production, indicating that these proteins may be involved in immune signaling. Collectively, our data show that Pp EC15 is conserved across P. pachyrhizi isolates and other economically important rust species and is involved in the suppression of plant basal defense responses. Understanding the role of Pp EC15 in P. pachyrhizi virulence will provide a foundation for designing targeted intervention strategies to generate rust-resistant crops.
Abstract Plant peroxisomes are highly dynamic organelles that mediate a suite of metabolic processes crucial to development. Peroxisomes in seeds/dark-grown seedlings and in photosynthetic tissues constitute two major subtypes of plant peroxisomes, which had been postulated to contain distinct primary biochemical properties. Multiple in-depth proteomic analyses had been performed on leaf peroxisomes, yet the major makeup of peroxisomes in seeds or dark-grown seedlings remained unclear. To compare the metabolic pathways of the two dominant plant peroxisomal subtypes and discover new peroxisomal proteins that function specifically during seed germination, we performed proteomic analysis of peroxisomes from etiolated Arabidopsis (Arabidopsis thaliana) seedlings. The detection of 77 peroxisomal proteins allowed us to perform comparative analysis with the peroxisomal proteome of green leaves, which revealed a large overlap between these two primary peroxisomal variants. Subcellular targeting analysis by fluorescence microscopy validated around 10 new peroxisomal proteins in Arabidopsis. Mutant analysis suggested the role of the cysteine protease RESPONSE TO DROUGHT21A-LIKE1 in β-oxidation, seed germination, and growth. This work provides a much-needed road map of a major type of plant peroxisome and has established a basis for future investigations of peroxisomal proteolytic processes to understand their roles in development and in plant interaction with the environment.
Abstract Drug-induced hepatotoxicity is one of the major reasons cited for drug withdrawal. Therefore, it is of extreme importance to detect human hepatotoxic candidates as early as possible during the drug development process. In this study, we aimed to enhance hepatocyte functions such as CYP gene expression in HepG2 cells, one of the most extensively used cell lines in evaluating hepatotoxicity of chemicals and drugs. We found that zebularine, a potent inhibitor of DNA methylation, remarkably upregulates the expression of CYP genes in HepG2 cells. In addition, we revealed that the upregulation of CYP gene expression by zebularine was mediated through the inhibition of both DNA methyltransferase 1 (DNMT1) and double-stranded RNA-dependent protein kinase (PKR). Furthermore, HepG2 cells treated with zebularine were more sensitive than control cells to drug toxicity. Taken together, our results show that zebularine may make HepG2 cells high-functioning and thus could be useful for evaluating the hepatotoxicity of chemicals and drugs speedily and accurately in in-vitro systems. The finding that zebularine upregulates CYP gene expression through DNMT1 and PKR modulation sheds light on the mechanisms controlling hepatocyte function and thus may aid in the development of new in-vitro systems using high-functioning hepatocytes.
A hallmark of multicellular organisms is their ability to maintain physiological homeostasis by communicating among cells, tissues, and organs. In plants, intercellular communication is largely dependent on plasmodesmata (PD), which are membrane-lined channels connecting adjacent plant cells. Upon immune stimulation, plants close PD as part of their immune responses. Here, we show that the bacterial pathogen Pseudomonas syringae deploys an effector protein, HopO1-1, that modulates PD function. HopO1-1 is required for P. syringae to spread locally to neighboring tissues during infection. Expression of HopO1-1 in Arabidopsis (Arabidopsis thaliana) increases the distance of PD-dependent molecular flux between neighboring plant cells. Being a putative ribosyltransferase, the catalytic activity of HopO1-1 is required for regulation of PD. HopO1-1 physically interacts with and destabilizes the plant PD-located protein PDLP7 and possibly PDLP5. Both PDLPs are involved in bacterial immunity. Our findings reveal that a pathogenic bacterium utilizes an effector to manipulate PD-mediated host intercellular communication for maximizing the spread of bacterial infection.
Peroxisomes are eukaryotic organelles with crucial functions in development. Plant peroxisomes participate in various metabolic processes, some of which are co-operated by peroxisomes and other organelles, such as mitochondria and chloroplasts. Defining the complete picture of how these essential organelles divide and proliferate will be instrumental in understanding how the dynamics of peroxisome abundance contribute to changes in plant physiology and development. Research in Arabidopsis thaliana has identified several evolutionarily conserved major components of the peroxisome division machinery, including five isoforms of PEROXIN11 proteins (PEX11), two dynamin-related proteins (DRP3A and DRP3B) and two FISSION1 proteins (FIS1A/BIGYIN and FIS1B). Recent studies in our laboratory have also begun to uncover plant-specific factors. DRP5B is a dual-localized protein that is involved in the division of both chloroplasts and peroxisomes, representing an invention of the plant/algal lineage in organelle division. In addition, PMD1 (peroxisomal and mitochondrial division 1) is a plant-specific protein tail anchored to the outer surface of peroxisomes and mitochondria, mediating the division and/or positioning of these organelles. Lastly, light induces peroxisome proliferation in dark-grown Arabidopsis seedlings, at least in part, through activating the PEX11b gene. The far-red light receptor phyA (phytochrome A) and the transcription factor HYH (HY5 homologue) are key components in this signalling pathway. In summary, pathways for the division and proliferation of plant peroxisomes are composed of conserved and plant-specific factors. The sharing of division proteins by peroxisomes, mitochondria and chloroplasts is also suggesting possible co-ordination in the division of these metabolically associated plant organelles.
'/%3e%3cuse xlink:href='%23a' transform='rotate(60)'/%3e%3ccircle r='17' fill='%23000095'/%3e%3ccircle r='15' fill='%23fff'/%3e%3c/svg%3e)