Identification of QTL associated with resistance to Phytophthora fruit rot in cucumber (Cucumis sativus L.).

Phytophthora fruit rot (PFR) caused by the soilborne oomycete pathogen, Phytophthora capsici, can cause severe yield loss in cucumber. With no resistant variety available, genetic resources are needed to develop resistant varieties. The goal of this work was to identify quantitative trait loci (QTL) associated with resistance to PFR using multiple genomic approaches and populations. Two types of resistances have been identified: age-related resistance (ARR) and young fruit resistance. ARR occurs at 12-16 days post pollination (dpp), coinciding with the end of exponential fruit growth. A major QTL for ARR was discovered on chromosome 3 and a candidate gene identified based on comparative transcriptomic analysis. Young fruit resistance, which is observed during the state of rapid fruit growth prior to commercial harvest, is a quantitative trait for which multiple QTL were identified. The largest effect QTL, qPFR5.1, located on chromosome 5 was fine mapped to a 1-Mb region. Genome-wide association studies (GWAS) and extreme-phenotype genome-wide association study (XP-GWAS) for young fruit resistance were also performed on a cucumber core collection representing > 96% of the genetic diversity of the USDA cucumber germplasm. Several SNPs overlapped with the QTL identified from QTL-seq analysis on biparental populations. In addition, novel SNPs associated with the resistance were identified from the germplasm. The resistant alleles were found mostly in accessions from India and South Asia, the center of diversity for cucumber. The results from this work can be applied to future disease resistance studies and marker-assisted selection in breeding programs.

PTC124 Rescues Nonsense Mutation of Two Tumor Suppressor Genes NOTCH1 and FAT1 to Repress HNSCC Cell Proliferation.

(1) Background: PTC124 (Ataluren) is an investigational drug for the treatment of nonsense mutation-mediated genetic diseases. With the exception of the TP53 tumor suppressor gene, there has been little research on cancers with nonsense mutation. By conducting a database search, we found that another two tumor suppressor genes, NOTCH1 and FAT1, have a high nonsense mutation rate in head and neck squamous cell carcinoma (HNSCC). PTC124 may re-express the functional NOTCH1 or FAT1 in nonsense mutation NOTCH1 or FAT1 in HSNCC (2) Methods: DOK (with NOTCH1 Y550X) or HO-1-u-1 (with FAT1 E378X) HNSCC cells were treated with PTC124, and the NOTCH1 or FAT1 expression, cell viability, and NOTCH1- or FAT1-related downstream gene profiles were assayed. (3) Results: PTC124 was able to induce NOTCH1 or FAT1 expression in DOK and HO-1-u-1 cells. PTC124 was able to upregulate NOTCH downstream genes HES5, AJUBA, and ADAM10 in DOK cells. PTC124 enhanced DDIT4, which is under the control of the FAT1-YAP1 pathway, in HO-1-u-1 cells. FLI-06 (a NOTCH signaling inhibitor) reversed PTC124-mediated cell growth inhibition in DOK cells. PTC124 could reverse TT-10 (a YAP signaling activator)-mediated HO-1-u-1 cell proliferation. (4) Conclusions: PTC124 can rescue nonsense mutation of NOTCH1 and FAT1 to repress HNSCC cell proliferation.

Mitochondrial oxidative stress in the tumor microenvironment and cancer immunoescape: foe or friend?

The major concept of "oxidative stress" is an excess elevated level of reactive oxygen species (ROS) which are generated from vigorous metabolism and consumption of oxygen. The precise harmonization of oxidative stresses between mitochondria and other organelles in the cell is absolutely vital to cell survival. Under oxidative stress, ROS produced from mitochondria and are the major mediator for tumorigenesis in different aspects, such as proliferation, migration/invasion, angiogenesis, inflammation, and immunoescape to allow cancer cells to adapt to the rigorous environment. Accordingly, the dynamic balance of oxidative stresses not only orchestrate complex cell signaling events in cancer cells but also affect other components in the tumor microenvironment (TME). Immune cells, such as M2 macrophages, dendritic cells, and T cells are the major components of the immunosuppressive TME from the ROS-induced inflammation. Based on this notion, numerous strategies to mitigate oxidative stresses in tumors have been tested for cancer prevention or therapies; however, these manipulations are devised from different sources and mechanisms without established effectiveness. Herein, we integrate current progress regarding the impact of mitochondrial ROS in the TME, not only in cancer cells but also in immune cells, and discuss the combination of emerging ROS-modulating strategies with immunotherapies to achieve antitumor effects.

P63 and P73 Activation in Cancers with p53 Mutation.

The members of the p53 family comprise p53, p63, and p73, and full-length isoforms of the p53 family have a tumor suppressor function. However, p53, but not p63 or p73, has a high mutation rate in cancers causing it to lose its tumor suppressor function. The top and second-most prevalent p53 mutations are missense and nonsense mutations, respectively. In this review, we discuss possible drug therapies for nonsense mutation and a missense mutation in p53. p63 and p73 activators may be able to replace mutant p53 and act as anti-cancer drugs. Herein, these p63 and p73 activators are summarized and how to improve these activator responses, particularly focusing on p53 gain-of-function mutants, is discussed.

Surface Modification of γ-Al2O3 Nanoparticles Using Conductive Polyaniline Doped by Dodecylbenzene Sulfonic Acid.

In this study, electrically conductive PANDB/γ-Al2O3 core-shell nanocomposites were synthesized by surface modification of γ-Al2O3 nanoparticles using polyaniline doped with dodecylbenzene sulfonic acid. The PANDB/γ-Al2O3 core-shell nanocomposites were synthesized by in situ polymerization. Pure PANDB and the PANDB/γ-Al2O3 core-shell nanocomposites were characterized using Fourier transform infrared spectroscopy, ultraviolet-visible spectroscopy, transmission electron microscopy, field emission scanning electron microscopy, and measurement of a four-point probe. The conductivity of the PANDB/γ-Al2O3 core-shell nanocomposite was about 0.72 S/cm when the weight ratio of aniline/γ-Al2O3 was 3/1. The results showed that the conductivity of the PANDB/γ-Al2O3 core-shell nanocomposite decreased with increasing amounts of γ-Al2O3 nanoparticles. The transmission electron microscopy results indicated that the γ-Al2O3 nanoparticles were thoroughly coated with PANDB to form a core-shell structure. Transmission electron microscopy and field emission scanning electron microscopy images of the conductive PANDB/γ-Al2O3 core-shell nanocomposites also showed that the thickness of the PANDB layer decreased as the amount of γ-Al2O3 was increased.

Impacts of phosphatidylglycerol on plastid gene expression and light induction of nuclear photosynthetic genes.

Phosphatidylglycerol (PG) is the only major phospholipid in the thylakoid membrane of chloroplasts. PG is essential for photosynthesis, and loss of PG in Arabidopsis thaliana results in severe defects of growth and chloroplast development, with decreased chlorophyll accumulation, impaired thylakoid formation, and down-regulation of photosynthesis-associated genes encoded in nuclear and plastid genomes. However, how the absence of PG affects gene expression and plant growth remains unclear. To elucidate this mechanism, we investigated transcriptional profiles of a PG-deficient Arabidopsis mutant pgp1-2 under various light conditions. Microarray analysis demonstrated that reactive oxygen species (ROS)-responsive genes were up-regulated in pgp1-2. However, ROS production was not enhanced in the mutant even under strong light, indicating limited impacts of photooxidative stress on the defects of pgp1-2. Illumination to dark-adapted pgp1-2 triggered down-regulation of photosynthesis-associated nuclear-encoded genes (PhANGs), while plastid-encoded genes were constantly suppressed. Overexpression of GOLDEN2-LIKE1 (GLK1), a transcription factor gene regulating chloroplast development, in pgp1-2 up-regulated PhANGs but not plastid-encoded genes along with chlorophyll accumulation. Our data suggest a broad impact of PG biosynthesis on nuclear-encoded genes partially via GLK1 and a specific involvement of this lipid in plastid gene expression and plant development.

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.

Totally tubeless single access tract mini-percutaneous nephrolithotripsy in treatment of large burden > 2-cm and/or complex renal stones: a case series of 62 patients.

BACKGROUND: Limited literature has focused on the use of totally tubeless mini-percutaneous nephrolithotomy (PCNL) for the treatment of large renal stones. We present our findings of treating patients with large and/or complex renal stones using single renal access totally tubeless mini-PCNL. METHODS: From March 2018 to May 2021, 62 consecutive cases in which single tract totally tubeless mini-PCNL was used to treat complex renal stones were enrolled, all with calculi > 2 cm. All procedure of puncture and dilation were guided by fluoroscope. The complexity of stones was assessed according to the Guy's Scoring System (GSS). The surgical duration, length of hospital stay, analgesia requirement, stone-free rate, and perioperative morbidity were assessed. RESULTS: The mean preoperative stone burden was 36.69 ± 19.76 mm (above 2 cm in all cases), mean surgical duration was 61.93 ± 40.84 min (range 15-180 min), and mean hematocrit reduction was 4.67 ± 2.83%. Postoperative Nalbuphine was used in 6 patients. The mean length of stay was 2.46 ± 1.19 days (range 2-8 days), and the postoperative stone-free rate was 83.9% (52/62), and 87.1% (54/62) after auxiliary ESWL. The overall complication rate was 14.5%, the majority of complications being postoperative transient fever. CONCLUSION: For the treatment of large bursen > 2 cm and/or complex renal stones, totally tubeless single tract mini-PCNL ensures a feasible SFR, low morbidity and short hospital stay. According to the low complication rate in our study, the totally tubeless manner was not associated with an increased risk of postoperative morbidity, and patients benefited from decreased postoperative analgesics use.

Crucial factors of the built environment for mitigating carbon emissions.

Global warming and environmental changes are becoming increasingly threatened by carbon emissions, especially in urban areas. Low-carbon cities have the co-benefits of mediating environmental threats and lowering carbon emissions. However, the direct and indirect pathways and effects between the built environment and carbon emissions remain unclear, limiting low-carbon city development. Therefore, this study used partial least squares (PLS) modeling and urban-scale data from nineteen counties in Taiwan to identify the crucial effects and pathways affecting carbon emissions. The model considered the impacts of the characteristics of urban form (i.e., density, land mix, city size, urban sprawl, and jobs-housing balance), urban function (i.e., industrial and commercial levels), urban transportation, and urban greening on carbon emissions. The results reveal that minimizing city size, urban sprawl, industrial level, and transportation status, and maximizing density, land mix, commercial levels, and urban green coverage could reduce carbon emissions. This is the first study to apply PLS modeling to identify variable pathways and evaluate both direct and indirect effects of built environment characteristics on carbon emissions. Findings demonstrated that appropriate urban policies and planning, such as compact cities, green cities, or transit-oriented development, might lower carbon emissions and thus further serve as useful strategies for building low-carbon cities.

Morphological and Genetic Diversity of Cucumber (Cucumis sativus L.) Fruit Development.

Cucumber (Cucumis sativus L.) fruits, which are eaten at an immature stage of development, can vary extensively in morphological features such as size, shape, waxiness, spines, warts, and flesh thickness. Different types of cucumbers that vary in these morphological traits are preferred throughout the world. Numerous studies in recent years have added greatly to our understanding of cucumber fruit development and have identified a variety of genetic factors leading to extensive diversity. Candidate genes influencing floral organ establishment, cell division and cell cycle regulation, hormone biosynthesis and response, sugar transport, trichome development, and cutin, wax, and pigment biosynthesis have all been identified as factors influencing cucumber fruit morphology. The identified genes demonstrate complex interplay between structural genes, transcription factors, and hormone signaling. Identification of genetic factors controlling these traits will facilitate breeding for desired characteristics to increase productivity, improve shipping, handling, and storage traits, and enhance consumer-desired qualities. The following review examines our current understanding of developmental and genetic factors driving diversity of cucumber fruit morphology.

Synthesis and Characterization of Conducting PANDB/χ-Al2O3 Core-Shell Nanocomposites by In Situ Polymerization.

Polyaniline doped with dodecylbenzenesulfonic acid/χ-aluminum oxide (PANDB/χ-Al2O3) conducting core-shell nanocomposites was synthesized via an in situ polymerization method in this study. PANDB was synthesized in the presence of dodecylbenzenesulfonic acid (DBSA), which functioned as a dopant and surfactant. The electrical conductivity of the conducting PANDB/χ-Al2O3 core-shell nanocomposite was approximately 1.7 × 10-1 S/cm when the aniline/χ-Al2O3 (AN/χ-Al2O3) weight ratio was 1.5. The transmission electron microscopy (TEM) results indicated that the χ-Al2O3 nanoflakes were thoroughly coated by PANDB to form the core-shell (χ-Al2O3-PANDB) structure. The TEM and field-emission scanning electron microscopy (FE-SEM) images of the conducting PANDB/χ-Al2O3 core-shell nanocomposites also indicated that the thickness of the PANDB layer (shell) could be increased as the weight ratio of AN/χ-Al2O3 was increased. In this study, the optimum weight ratio of AN/χ-Al2O3 was identified as 1.5. The conducting PANDB/χ-Al2O3 core-shell nanocomposite was then blended with water-based polyurethane (WPU) to form a conducting WPU/PANDB/χ-Al2O3 blend film. The resulting blend film has promising antistatic and electrostatic discharge (ESD) properties.

Quantitative High-Throughput, Real-Time Bioassay for Plant Pathogen Growth in vivo.

Effective assessment of pathogen growth can facilitate screening for disease resistance, mapping of resistance loci, testing efficacy of control measures, or elucidation of fundamental host-pathogen interactions. Current methods are often limited by subjective assessments, inability to detect pathogen growth prior to appearance of symptoms, destructive sampling, or limited capacity for replication and quantitative analysis. In this work we sought to develop a real-time, in vivo, high-throughput assay that would allow for quantification of pathogen growth. To establish such a system, we worked with the broad host-range, highly destructive, soil-borne oomycete pathogen, Phytophthora capsici. We used an isolate expressing red fluorescence protein (RFP) to establish a microtiter plate, real-time assay to quantify pathogen growth in live tissue. The system was successfully used to monitor P. capsici growth in planta on cucumber (Cucumis sativus) fruit and pepper (Capsicum annuum) leaf samples in relation to different levels of host susceptibility. These results demonstrate usefulness of the method in different species and tissue types, allowing for highly replicated, quantitative time-course measurements of pathogen growth in vivo. Analyses of pathogen growth during initial stages of infection preceding symptom development show the importance of very early stages of infection in determining disease outcome, and provide insight into points of inhibition of pathogen growth in different resistance systems.

Developmentally regulated activation of defense allows for rapid inhibition of infection in age-related resistance to Phytophthora capsici in cucumber fruit.

BACKGROUND: Age-related resistance (ARR) is a developmentally regulated phenomenon conferring resistance to pathogens or pests. Although ARR has been observed in several host-pathogen systems, the underlying mechanisms are largely uncharacterized. In cucumber, rapidly growing fruit are highly susceptible to Phytophthora capsici but become resistant as they complete exponential growth. We previously demonstrated that ARR is associated with the fruit peel and identified gene expression and metabolomic changes potentially functioning as preformed defenses. RESULTS: Here, we compare the response to infection in fruit at resistant and susceptible ages using microscopy, quantitative bioassays, and weighted gene co-expression analyses. We observed strong transcriptional changes unique to resistant aged fruit 2-4 h post inoculation (hpi). Microscopy and bioassays confirmed this early response, with evidence of pathogen death and infection failure as early as 4 hpi and cessation of pathogen growth by 8-10 hpi. Expression analyses identified candidate genes involved in conferring the rapid response including those encoding transcription factors, hormone signaling pathways, and defenses such as reactive oxygen species metabolism and phenylpropanoid biosynthesis. CONCLUSION: The early pathogen death and rapid defense response in resistant-aged fruit provide insight into potential mechanisms for ARR, implicating both pre-formed biochemical defenses and developmentally regulated capacity for pathogen recognition as key factors shaping age-related resistance.

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.

High-Resolution Crystal Structure of Arabidopsis FLOWERING LOCUS T Illuminates Its Phospholipid-Binding Site in Flowering.

Arabidopsis FLOWERING LOCUS T (FT) is a pivotal component of florigen, a long-range mobile flowering signal. Here, we determined the 1.0 Å-resolution crystal structure of FT, a significantly higher-resolution crystal structure of FT than previously reported one (2.6 Å). The present crystallographic studies revealed 4 alternative configurations with the precise location of the surrounding water molecules. Using this structural data, computational docking simulation predicted the putative binding sites for phosphatidylcholine (PC), an endogenous ligand that interacts with FT to modulate flowering time. In vitro reconstitution of the lipid-protein interaction showed that mutations at two of the predicted sites significantly compromised the lipid binding ability of FT. In planta, one of the mutant FT proteins significantly affected FT function in flowering, emphasizing the involvement of PC binding in modulating FT function. Our structural, biochemical, and transgenic analyses reveal the molecular mechanism of PC binding in FT-mediated flowering time control.

Arabidopsis CHOLINE/ETHANOLAMINE KINASE 1 (CEK1) is a primary choline kinase localized at the endoplasmic reticulum (ER) and involved in ER stress tolerance.

Choline kinase catalyzes the initial reaction step of choline metabolism that produces phosphocholine, a prerequisite for the biosynthesis of a primary phospholipid phosphatidylcholine. However, the primary choline kinase and its role in plant growth remained elusive in seed plants. Here, we showed that Arabidopsis CHOLINE/ETHANOLAMINE KINASE 1 (CEK1) encodes functional CEK that prefers choline than ethanolamine as a substrate in vitro and affects contents of choline and phosphocholine but not phosphatidylcholine in vivo. CEK1 is localized at endoplasmic reticulum (ER); upon tunicamycin-induced ER stress, a null mutant of CEK1 showed hypersensitive phenotype in seedlings, albeit with no enhanced choline kinase activity. Our results demonstrate that CEK1 is a primary ER-localized choline kinase in vivo that is required for ER stress tolerance possibly through the modulation of choline metabolites.

A Methyltransferase Trio Essential for Phosphatidylcholine Biosynthesis and Growth.

Phosphatidylcholine (PC) is a primary class of membrane lipids in most eukaryotes. In plants, the primary PC biosynthetic pathway and its role in plant growth and development remain elusive due to lack of a mutant model with substantially decreased PC content. Recently, a double mutant of Arabidopsis (Arabidopsis thaliana) PHOSPHO-BASE N-METHYLTRANSFERASE 1 (PMT1) and PMT3 was reported with reduced PC content and defective plant growth. However, residual PC content as well as the nonlethal phenotype of the mutant suggests an additional enzyme contributes to PC biosynthesis. In this article, we report on the role of three PMTs in PC biosynthesis and plant development, with a focus on PMT2. PMT2 had the highest expression level among the three PMTs, and it was highly expressed in roots. The pmt1 pmt2 double mutant enhanced the defects in root growth, cell viability, and PC content of pmt1, suggesting that PMT2 functions together with PMT1 in roots. Chemical inhibition of PMT activity in wild-type roots reproduced the short root phenotype observed in pmt1 pmt2, suggesting that PMT1 and PMT2 are the major PMT isoforms in roots. In shoots, pmt1 pmt2 pmt3 enhanced the phenotype of pmt1 pmt3, showing seedling lethality and further reduced PC content without detectable de novo PC biosynthesis. These results suggest that PMTs catalyze an essential reaction step in PC biosynthesis and that the three PMTs have differential tissue-specific functions in PC biosynthesis and plant growth.

Detection of Particulate Matter of Size 2.5 µm with a Surface-Acoustic-Wave Sensor Combined with a Cyclone Separator.

A device to monitor particulate matter of size 2.5 µm (PM2.5) that has been designed and developed includes a surface-acoustic-wave sensor operating in a shear horizontal mode (SH-SAW) combined with a cyclone separator. In our tests, aerosols generated as incense smoke were first separated and sampled inside a designed cyclone separator; the sampled PM2.5 was then introduced into the sensing area of an SH-SAW sensor for detection. The use of microcentrifuge tubes as a cyclone separator effectively decreases the size and power consumption of the device; the SAW sensor in a well design and operating at 122 MHz was fabricated with MEMS techniques. After an explanation of the design of the cyclone separator, a simulation of the efficiency and the SAW sensor detection are discussed. A microcentrifuge tube (volume 0.2 mL, inlet and outlet diameters 0.5 mm) as a separator has separation cutoff diameters 50% (d50) at 2.5 µm; the required rate of volumetric flow at the inlet is 0.125 LPM, according to simulation with computational fluid dynamics (CFD) software; the surface-acoustic-wave (SAW) sensor exhibits sensitivity approximately 9 Hz/ng; an experiment for PM2.5 detection conducted with the combined device shows a strong positive linear correlation with a commercial aerosol monitor. The limit of detection (LOD) is 11 µg/m³ with sample time 160 s and total detection duration about 5 min.

A pair of phospho-base methyltransferases important for phosphatidylcholine biosynthesis in Arabidopsis.

Phosphatidylcholine (PtdCho) is a predominant membrane lipid class in eukaryotes. Phospho-base N-methyltransferase (PMT) catalyzes a critical step in PtdCho biosynthesis. However, in Arabidopsis thaliana, the discovery of involvement of the specific PMT isoform in PtdCho biosynthesis remains elusive. Here, we show that PMT1 and PMT3 redundantly play an essential role in phosphocholine (PCho) biosynthesis, a prerequisite for PtdCho production. A pmt1 pmt3 double mutant was devoid of PCho, which affected PtdCho biosynthesis in vivo, showing severe growth defects in post-embryonic development. PMT1 and PMT3 were both highly expressed in the vasculature. The pmt1 pmt3 mutants had specifically affected leaf vein development and showed pale-green seedlings that were rescued by exogenous supplementation of PCho. We suggest that PMT1 and PMT3 are the primary enzymes for PCho biosynthesis and are involved in PtdCho biosynthesis and vascular development in Arabidopsis seedlings.

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.