Organoiridium Complexes Enhance Cellular Defense Against Reactive Aldehydes Species.

Although reactive aldehyde species (RASP) are associated with the pathogenesis of many major diseases, there are currently no clinically approved treatments for RASP overload. Conventional aldehyde detox agents are stoichiometric reactants that get consumed upon reacting with their biological targets, which limits their therapeutic efficiency. To achieve longer-lasting detoxification effects, small-molecule intracellular metal catalysts (SIMCats) were used to protect cells by converting RASP into non-toxic alcohols. It was shown that SIMCats were significantly more effective in lowering cell death from the treatment with 4-hydroxynon-2-enal than aldehyde scavengers over a 72 h period. Studies revealed that SIMCats reduced the aldehyde accumulation in cells exposed to the known RASP inducer arsenic trioxide. This work demonstrates that SIMCats offer unique benefits over stochiometric agents, potentially providing new ways to combat diseases with greater selectivity and efficiency than existing approaches.

Fluorescent half-sandwich iridium picolinamidate complexes for in-cell visualization.

In this work, we report on the development of fluorescent half-sandwich iridium complexes using a fluorophore attachment strategy. These constructs consist of pentamethylcyclopentadienyl (Cp*) iridium units ligated by picolinamidate donors conjugated to green-emitting boron-dipyrromethene (bodipy) dyes. Reaction studies in H2O/THF mixtures showed that the fluorescent Ir complexes were active as catalysts for transfer hydrogenation, with activities similar to that of their non-fluorescent counterparts. The iridium complexes were taken up by NIH-3T3 mouse fibroblast cells, with 50% inhibition concentrations ranging from ~20-70 µM after exposure for 3 h. Visualization of the bodipy-functionalized Ir complexes in cells using fluorescence microscopy revealed that they were localized in the mitochondria and lysosome but not the nucleus. These results indicate that our fluorescent iridium complexes could be useful for future biological studies requiring intracellular catalyst tracking.

Phosphate starvation-inducible GLYCEROPHOSPHODIESTER PHOSPHODIESTERASE6 is involved in Arabidopsis root growth.

Plants that are starved of phosphate trigger membrane lipid remodeling, which hydrolyses phospholipids and presumably allows their phosphate to be utilized, whilst replacing them with galactolipids to maintain the integrity of the membrane system. In addition to the two concurrent pathways of phospholipid hydrolysis by phospholipases C and D that have already been established, an emerging third pathway has been proposed that includes a reaction step catalysed by glycerophosphodiester phosphodiesterases (GDPDs). However, its functional involvement in phosphate-starved plants remains elusive. Here, we show that Arabidopsis GDPD6 is a functional isoform responsible for glycerophosphocholine hydrolysis in vivo. Overexpression of GDPD6 promoted root growth whilst gdpd6 mutants showed impaired root growth under phosphate starvation, and this defect was rescued by supplementing with the reaction product glycerol 3-phosphate but not with choline. Since GDPD6 is induced by phosphate starvation, we suggest that GDPD6 might be involved in root growth via the production of glycerol 3-phosphate in phosphate-starved plants.

The phospho-base N-methyltransferases PMT1 and PMT2 produce phosphocholine for leaf growth in phosphorus-starved Arabidopsis.

Phosphorus (P) is an essential nutrient for plants. Membrane lipid remodeling is an adaptive mechanism for P-starved plants that replaces membrane phospholipids with non-P galactolipids, presumably to retrieve scarce P sources and maintain membrane integrity. Whereas metabolic pathways to convert phospholipids to galactolipids are well-established, the mechanism by which phospholipid biosynthesis is involved in this process remains elusive. Here, we report that phospho-base N-methyltransferases 1 and 2 (PMT1 and PMT2), which convert phosphoethanolamine to phosphocholine (PCho), are transcriptionally induced by P starvation. Shoots of seedlings of pmt1 pmt2 double mutant showed defective growth upon P starvation; however, membrane lipid profiles were unaffected. We found that P-starved pmt1 pmt2 with defective leaf growth had reduced PCho content, and the growth defect was rescued by exogenous supplementation of PCho. We propose that PMT1 and PMT2 are induced by P starvation to produce PCho mainly for leaf growth maintenance, rather than for phosphatidylcholine biosynthesis, in membrane lipid remodeling.

Non-specific phospholipases C2 and C6 redundantly function in pollen tube growth via triacylglycerol production in Arabidopsis.

Non-specific phospholipase Cs (NPCs) are responsible for membrane lipid remodeling that involves hydrolysis of the polar head group of membrane phospholipids. Arabidopsis NPC2 and NPC6 are essential in gametogenesis, but their underlying role in the lipid remodeling remains elusive. Here, we show that these NPCs are required for triacylglycerol (TAG) production in pollen tube growth. NPC2 and NPC6 are highly expressed in developing pollen tubes and are localized at the endoplasmic reticulum. Mutants of NPC2 and NPC6 showed reduced rate of pollen germination, length of pollen tube and amount of lipid droplets (LDs). Overexpression of NPC2 or NPC6 induced LD accumulation, which suggests that these NPCs are involved in LD production. Furthermore, mutants defective in the biosynthesis of TAG, a major component of LDs, showed defective pollen tube growth. These results suggest that NPC2 and NPC6 are essential in gametogenesis for a role in hydrolyzing phospholipids and producing TAG required for pollen tube growth. Thus, lipid remodeling from phospholipids to TAG during pollen tube growth represents an emerging role for the NPC family in plant developmental control.

Non-specific phospholipase C (NPC): an emerging class of phospholipase C in plant growth and development.

Non-specific phospholipase C (NPC) is a novel class of phospholipase C found only in bacteria and higher plants. NPC hydrolyzes major phospholipid classes such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE) to produce diacylglycerol (DAG) and a corresponding phosphate-containing polar head group. Originally known as a toxin in certain bacteria to invade the host cell, this class of phospholipase has been well-investigated in bacteriology. Since the first discovery of eukaryotic NPC in Arabidopsis in 2005, this emerging class of phospholipase has received greater attention in plant biology in elucidating the biochemical characteristics and physiological function in the context of plant growth regulation and stress response. Particularly in the last few years, there has been significant progress made in understanding the fundamental character of 6 NPC isoforms in Arabidopsis, as well as novel function in other plant models. Now that research with plant NPC is entering into a new phase, this review aims to summarize recent progress in plant NPC along with some future perspectives.

Correction to: Non-specific phospholipase C (NPC): an emerging class of phospholipase C in plant growth and development.

In the original publication of the article, values of "Genes" NPC6 was out of order in Table 1.

The Four Arabidopsis Choline/Ethanolamine Kinase Isozymes Play Distinct Roles in Metabolism and Development.

Phosphatidylcholine and phosphatidylethanolamine are two major phospholipid classes in eukaryotes. Each biosynthesis pathway starts with the phosphorylation of choline (Cho) or ethanolamine (Etn) catalyzed by either choline or ethanolamine kinase (CEK). Arabidopsis contains four CEK isoforms, but their isozyme-specific roles in metabolism and development are poorly described. Here, we showed that these four CEKs have distinct substrate specificities in vitro. While CEK1 and CEK2 showed substrate preference for Cho over Etn, CEK3 and CEK4 had clear substrate specificity for Cho and Etn, respectively. In vivo, CEK1, CEK2, and CEK3 exhibited kinase activity for Cho but not Etn, although the latter two isoforms showed rather minor contributions to total Cho kinase activity in both shoots and roots. The knockout mutants of CEK2 and CEK3 both affected root growth, and these isoforms had nonoverlapping cell-type-specific expression patterns in the root meristematic zone. In-depth phenotype analysis, as well as chemical and genetic complementation, revealed that CEK3, a Cho-specific kinase, is involved in cell elongation during root development. Phylogenetic analysis of CEK orthologs in Brassicaceae species showed evolutionary divergence between Etn kinases and Cho kinases. Collectively, our results demonstrate the distinct roles of the four CEK isoforms in Cho/Etn metabolism and plant development.

Non-specific phospholipases C, NPC2 and NPC6, are required for root growth in Arabidopsis.

Mutants in lipid metabolism often show a lethal phenotype during reproduction that prevents investigating a specific role of the lipid during different developmental processes. We focused on two non-specific phospholipases C, NPC2 and NPC6, whose double knock-out causes a gametophyte-lethal phenotype. To investigate the role of NPC2 and NPC6 during vegetative growth, we produced transgenic knock-down mutant lines that circumvent the lethal effect during gametogenesis. Despite no defect observed in leaves, root growth was significantly retarded, with abnormal cellular architecture in root columella cells. Furthermore, the short root phenotype was rescued by exogenous supplementation of phosphocholine, a product of non-specific phospholipase C (NPC) -catalyzed phosphatidylcholine hydrolysis. The expression of phospho-base N-methyltransferase 1 (PMT1), which produces phosphocholine and is required for root growth, was induced in the knock-down mutant lines and was attenuated after phosphocholine supplementation. These results suggest that NPC2 and NPC6 may be involved in root growth by producing phosphocholine via metabolic interaction with a PMT-catalyzed pathway, which highlights a tissue-specific role of NPC enzymes in vegetative growth beyond the gametophyte-lethal phenotype.

Expression Profiles of 2 Phosphate Starvation-Inducible Phosphocholine/Phosphoethanolamine Phosphatases, PECP1 and PS2, in Arabidopsis.

Phosphorus is essential for plant viability. Phosphate-starved plants trigger membrane lipid remodeling to replace membrane phospholipids by non-phosphorus galactolipids presumably to acquire scarce phosphate source. Phosphoethanolamine/phosphocholine phosphatase 1 (PECP1) and phosphate starvation-induced gene 2 (PS2) belong to an emerging class of phosphatase induced by phosphate starvation and dephosphorylates phosphocholine and phosphoethanolamine (PEtn) in vivo. However, detailed spatiotemporal expression pattern as well as subcellular localization has not been investigated yet. Here, by constructing transgenic plants harboring functional translational promoter-reporter fusion system, we showed the expression pattern of PECP1 and PS2 in different tissues and in response to phosphate starvation. Besides, the Venus fluorescent reporter revealed that both are localized at the ER. Characterization of transgenic plants that overexpress PECP1 or PS2 showed that their activity toward PEtn may be different in vivo. We suggest that PECP1 and PS2 are ER-localized phosphatases that show similar expression pattern yet have a distinct substrate specificity in vivo.

A pair of nonspecific phospholipases C, NPC2 and NPC6, are involved in gametophyte development and glycerolipid metabolism in Arabidopsis.

Phospholipases play crucial roles in plant membrane lipid homeostasis. Nonspecific phospholipase C (NPCs) establish a unique class of phospholipases found only in plants and certain bacteria. Here, we show that two previously uncharacterized NPC isoforms, NPC2 and NPC6, are required for male and female gametophyte development in Arabidopsis. Double mutant plants of npc2-1 npc6-2 could not be retrieved because npc2-1 npc6-2 ovule and pollen development is affected. Genetic complementation, reciprocal crossing and microscope observation of npc2-1/- npc6-2/+ and npc2-1/+ npc6-2/- plants suggest that NPC2 and NPC6 are redundant and are required for normal gametophyte development. Both NPC2 and NPC6 proteins are localized to the plastids. Promoter-GUS assays in transgenic Arabidopsis revealed that NPC2 and NPC6 are preferentially expressed in floral organs rather than in leaves. In vitro enzyme assays showed that NPC2 and NPC6 hydrolyze phosphatidylcholine and phosphatidylethanolamine, but not phosphatidate, being consistent with the reported substrate selectivity of NPCs. The amounts of phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol were increased in buds but not in flowers of npc2-1/- npc6-2/+ and npc2-1/+ npc6-2/- plants, presumably due to reduced phospholipid hydrolysis activity in developing flowers. Our results demonstrate that NPC2 and NPC6 play crucial roles in gametogenesis during flower development.

Intracellular Chemistry: Integrating Molecular Inorganic Catalysts with Living Systems.

This concept article focuses on the rapid growth of intracellular chemistry dedicated to the integration of small-molecule metal catalysts with living cells and organisms. Although biological systems contain a plethora of biomolecules that can deactivate inorganic species, researchers have shown that small-molecule metal catalysts could be engineered to operate in heterogeneous aqueous environments. Synthetic intracellular reactions have recently been reported for olefin hydrogenation, hydrolysis/oxidative cleavage, azide-alkyne cycloaddition, allylcarbamate cleavage, C-C bond cross coupling, and transfer hydrogenation. Other promising targets for new biocompatible reaction discovery will also be discussed, with a special emphasis on how such innovations could lead to the development of novel technologies and chemical tools.

Intracellular Transfer Hydrogenation Mediated by Unprotected Organoiridium Catalysts.

In the present work, we show for the first time that the conversion of aldehydes to alcohols can be achieved using "unprotected" iridium transfer hydrogenation catalysts inside living cells. The reactions were observed in real time by confocal fluorescence microscopy using a Bodipy fluorogenic substrate. We propose that the reduced cofactor nicotinamide adenine dinucleotide (NADH) is a possible hydride source inside the cell based on studies using pyruvate as a cellular redox modulator. We expect that this biocompatible reductive chemistry will be broadly useful to practitioners working at the interface of chemistry and the life sciences.

Innocent But Deadly: Nontoxic Organoiridium Catalysts Promote Selective Cancer Cell Death.

We demonstrate that nontoxic organoiridum complexes can selectively chemosensitize cancer cells toward platinum antiproliferative agents. Treatment of human cancer cells (breast, colon, eye/retina, head/neck, lung, ovary, and blood) with the iridium chemosensitizers led to lowering of the 50 % growth inhibition concentration (IC50 ) of the Pt drug carboplatin by up to ∼30-50 %. Interestingly, non-cancer cells were mostly resistant to the chemosensitizing effects of the iridium complexes. Cell culture studies indicate that cancer cells that were administered with Ir show significantly higher reactive oxygen species concentrations as well as NAD+ /NADH ratios (oxidized vs. reduced nicotinamide adenine dinucleotide) than Ir-treated non-cancer cells. These biochemical changes are consistent with a catalytic transfer hydrogenation cycle involving the formation of iridium-hydride species from the reaction of the iridium catalysts with NADH and subsequent oxidation in air to generate hydrogen peroxide.