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Research ArticleBIOENERGETICS AND PHOTOSYNTHESIS
Towards an Analysis of the Rice Mitochondrial Proteome
Joshua L. Heazlewood, Katharine A. Howell, James Whelan, A. Harvey Millar
Joshua L. Heazlewood
Katharine A. Howell
A. Harvey Millar
Published May 2003. DOI: https://doi.org/10.1104/pp.102.018986
Copyright © 2003 American Society of Plant Biologists
Purified rice (Oryza sativa) mitochondrial proteins have been arrayed by isoelectric focusing/polyacrylamide gel electrophoresis (PAGE), by blue-native (BN) PAGE, and by reverse-phase high-performance liquid chromatography (LC) separation (LC-mass spectrometry [MS]). From these protein arrays, we have identified a range of rice mitochondrial proteins, including hydrophilic/hydrophobic proteins (grand average of hydropathicity = −1.27 to +0.84), highly basic and acid proteins (isoelectric point = 4.0–12.5), and proteins over a large molecular mass range (6.7–252 kD), using proteomic approaches. BN PAGE provided a detailed picture of electron transport chain protein complexes. A total of 232 protein spots from isoelectric focusing/PAGE and BN PAGE separations were excised, trypsin digested, and analyzed by tandem MS (MS/MS). Using this dataset, 149 of the protein spots (the products of 91 nonredundant genes) were identified by searching translated rice open reading frames from genomic sequence and six-frame translated rice expressed sequence tags. Sequence comparison allowed us to assign functions to a subset of 85 proteins, including many of the major function categories expected for this organelle. A further six spots were matched to rice sequences for which no specific function has yet been determined. Complete digestion of mitochondrial proteins with trypsin yielded a peptide mixture that was analyzed directly by reverse-phase LC via organic solvent elution from a C-18 column (LC-MS). These data yielded 170 MS/MS spectra that matched 72 sequence entries from open reading frame and expressed sequence tag databases. Forty-five of these were obtained using LC-MS alone, whereas 28 proteins were identified by both LC-MS and gel-based separations. In total, 136 nonredundant rice proteins were identified, including a new set of 23 proteins of unknown function located in plant mitochondria. We also report the first direct identification, to our knowledge, of PPR (pentatricopeptide repeat) proteins in the plant mitochondrial proteome. This dataset provides the first extensive picture, to our knowledge, of mitochondrial functions in a model monocot plant.
The ATP-synthesizing organelles of eukaryotic cells, mitochondria, can trace their origins back to an event in which one prokaryotic cell was engulfed by another cell to form a new cellular lineage containing two genomes. These genomes became dependent over time, and mitochondria lost the ability to be viable outside the host cell. A significant net transfer of genetic information occurred from the mitochondrial to the nuclear genome during this time. It is predicted that mitochondria now synthesize only a few percent of the proteins required for their function, with the majority of their proteins being encoded in the nucleus, translated in the cytosol, and transferred back to the mitochondria as protein precursors by means of targeting information in the protein sequence (Gray et al., 1999). Biochemical investigations have revealed that isolated plant mitochondria share many features with those from animals and fungi. However, plant mitochondria also contain additional components such as non-phosphorylating bypasses of the electron transport chain, specialized metabolite carriers, and enzymes involved in the synthesis of folate, lipoic acid, and vitamin C (Bartoli et al., 2000; Gueguen et al., 2000; Rebeille et al., 1997). Only a few of the nuclear genes encoding proteins that maintain these unique functions have been identified. The recent increased sequencing of plant genomes (Arabidopsis Genome Initiative, 2000; Goff et al., 2002; Yu et al., 2002) has encouraged the development of bioinformatic tools to predict the subcellular localization of putative plant protein sequences. Programs such as MitoProtII (Claros and Vincens, 1996), TargetP (Emanuelsson et al., 2000), and Predotar (http://www.inra.fr/Internet/ Produits/Predotar/) predict that thousands of nuclear-encoded plant proteins are destined for mitochondria, but the veracity of such predictions still awaits confirmation.
Several reports have recently attempted to identify the protein contents of plant mitochondria from Arabidopsis (Kruft et al., 2001; Millar et al., 2001) and pea (Pisum sativum; Bardel et al., 2002) using isoelectric focusing (IEF)/SDS-PAGE-based separation of proteins. These three reports each identified 40 to 90 proteins using this arraying technique coupled with identification using antibodies, N-terminal sequencing, matrix-assisted laser-desorption/ionization-time-of-flight (TOF) peptide mass fingerprinting, and collision-induced tandem mass spectrometry (MS/MS) spectra of single peptides. Both these species are dicots. Increasing evidence of important differences between monocot and dicot plant lineages at a molecular level have been reported (Qiu et al., 1999; Soltis et al., 1999; Daly et al., 2001). Differences in mitochondrial molecular function between monocots and dicots also have been highlighted recently (Fey and Marechal-Drouard, 1999; Thelen et al., 1999; Considine et al., 2002). Thus, we consider it important to consider the mitochondrial proteome in a model monocot species.
The gel array technique used in the dicot mitochondrial proteome reports has several limitations. In particular, very small (<10 kD) and very large (>100 kD) proteins, basic proteins (pI > 10), and hydrophobic proteins (grand average of hydropathicity [GRAVY] > 0.2) are poorly resolved by this method (Chevallet et al., 1998; Herbert et al., 1998; Santoni et al., 1999, 2000). Incremental improvements in chaotropic agents and nonionic detergents have improved the resolving power of classical denaturing two-dimensional electrophoresis (Chevallet et al., 1998; Herbert et al., 1998). However, a substantial protein set will probably always be excluded due to the physical limitations of this approach. Native gel electrophoresis coupled to second dimension SDS-PAGE provides a flexible solution to the hydrophobic protein exclusion problem. Many such proteins are not found exposed in biological membranes but are core, internalized components of multisubunit complexes. Maintenance of this complex structure avoids precipitation of hydrophobic components. Blue-native (BN) PAGE has been used previously to separate native protein complexes from mitochondria (Schagger et al., 1994). A subsequent denaturing SDS-PAGE dimension allows separation of individual components for analysis from plant mitochondria (Jansch et al., 1996; Werhahn and Braun, 2002). A different solution to the exclusion problem, with an even wider applicability, is non-gel-based separation of complex mixtures. These approaches often use liquid chromatography (LC)-based separations of peptide derived from whole-sample digests that are directly coupled to MS. One approach using a coupled strong cation affinity and reverse-phase C18 separation has been termed multidimensional protein identification technology and is being increasingly applied to a broad range of biological samples (Washburn et al., 2001).
To determine which putative proteins from the rice (Oryza sativa) genome sequence represent constitutively expressed proteins involved in mitochondrial function, we have used two-dimensional gel electrophoresis and LC analysis of purified rice mitochondria coupled with MS identification. Our aim was to identify proteins localized in rice mitochondria, compare and contrast protein display techniques, and identify novel mitochondrial proteins, thereby providing a substantial set of experimentally identified mitochondrial proteins from this model monocot plant.
Integrity and Function of Isolated Rice Mitochondria
The integrity of a subcellular proteome, such as that of mitochondria, is largely dependent on the purification of the isolated compartment away from other cellular contaminants. We have adapted a two-Percoll gradient density separation technique to the isolation of these organelles from dark-grown rice shoots that yields mitochondria largely free of contamination by cytosol, peroxisomes, plastids, and other membranes (Millar et al., 2001). We have assayed the integrity and function of these purified rice mitochondria to ensure that proteins were not being lost by rupture during isolation and that key respiratory functions were maintained. The outer membrane of mitochondrial samples was found to be 96% intact based on the latency of cytochrome c oxidase activity initiated by exogenously added cytochrome c (Table I). Whole-electron transport chain activities showed that O2consumption by the mitochondria could be supported by succinate, external NADH, or a combination of malate and pyruvate. O2 consumption supported by each of these substrates was significantly and transiently stimulated by the addition of ADP. These data suggested the presence and function of the electron transport chain, the F1F0-ATP synthase, the tricarboxylic acid cycle in the mitochondrial matrix, the integrity of the inner membrane, and the function of external rotenone-insensitive NADH dehydrogenase bypasses of complex I (Table I). Little or no respiration was maintained in the presence of the complex III inhibitor myxothiazol, showing that little or no alternative oxidase (AOX) was present in these mitochondria (Table I). This lack of AOX was also confirmed by addition of pyruvate and dithiothreitol to O2 electrode traces without effect after myxothiazol addition. It was further confirmed by the lack of immunodetectable bands using monoclonal antibodies raised against AOX (data not shown).
O2 consumption by isolated rice mitochondria following addition of different substrates, effectors, and inhibitors
IEF and BN Arraying of Rice Mitochondrial Proteins
To analyze the complex mixture of proteins present in rice mitochondria, these proteins were separated in gel arrays using the apparent pI and molecular mass of individual polypeptides and the apparent native molecular mass of protein complexes. The IEF/SDS-PAGE separation used the first two of these properties and revealed a set of approximately 250 protein spots (Fig. 1). A set of 145 of the more abundant spots from this set was excised and subjected to in-gel digestion followed by MS/MS-based analysis of the resultant peptides. This analysis lead to the identification of 80 of these protein spots as the products of 63 predicted gene sequences from rice (Table II).
IEF/SDS-PAGE separation of rice mitochondrial proteins (pI = 3–10). A total of 145 protein spots from this map were excised, digested, and analyzed by MS/MS. Eighty were identified, and these are numbered for comparison with text and Table II. Numbers on the horizontal axis are pI, and numbers on the vertical axis are apparent molecular mass (in kilodaltons).
Identification of proteins from purified rice mitochondria using MS/MS peptide spectra matched to rice ORFs translated by the Institute for Genomic Research (TIGR; XXXX.mXXXXX), TIGR-translated expressed sequence tags (ESTs, TCXXXXX), and rice NCBI entries
The BN SDS-PAGE uses a combination of native masses of complexes in the first dimension and the denatured masses of polypeptides in the second dimension (Fig. 2). This analysis was only performed on the membrane fraction from rice mitochondria that contains a large number of hydrophobic proteins that were unlikely to be soluble under IEF conditions. Protein staining revealed a series of large complexes of comigrating subunits that constitute the major electron transport chain complexes: complexes I, III, and V (Fig. 2). A set of approximately 100 spots was visible, a set of 89 spots was excised, and a subset of 57 spots from this set was identified as the products of 49 rice genes. There are several examples where a single protein sample contained peptides derived from two different and unrelated gene products; note spot 44 (cytochrome oxidase subunit I and malic enzyme), spot 57 (adrenodoxin and TIM17), and spot 42 (TOM9 and ATP synthase, 6 kD). In all these cases, the MS evidence for the presence of both proteins in very convincing. Because streaking of some proteins occurs during native gel electrophoresis, it is possible that the same proteins do exist more discretely elsewhere on the gel and that our identification point (i.e. the spot excision point) is just coincidently the point of overlap. Comparison of these identified sets from IEF/SDS-PAGE and BN SDS-PAGE revealed that there were 21 nonredundant gene matches in common, whereas together, they represent a combined nonredundant set of 91 gene products. A set of 18 well-known components of mitochondrial inner and outer membrane complexes was identified in the BN SDS-PAGE analysis that was not identified from the IEF/SDS-PAGE analysis (Table II).
BN SDS-PAGE separation of rice membrane mitochondrial proteins. A total of 89 protein spots from this map were excised, digested, and analyzed by MS/MS. Fifty-seven were identified, and these are numbered for comparison with text and Table II. Numbers on vertical axis are apparent molecular mass of protein subunits (kilodaltons), and annotations on horizontal axis are known mitochondrial protein complexes identified in this study.
Matching of MS/MS spectra from peptides from IEF/SDS-PAGE and BN SDS-PAGE spots to predicted gene products was performed using ProID software (Applied Biosystems, Sydney) with custom-made databases using TIGR rice open reading frame and EST data. Many of the rice proteins identified were not represented at National Center for Biotechnology Information; thus, Mascot on-line (http://www.matrixscience.com) could not be used routinely for analysis. ProID was used with mass accuracy cutoffs of peptide mass ± 0.15 and MS/MS ions ± 0.05, which are significantly more stringent than Mascot default settings. In most cases, more than three matching MS/MS spectra were identified by ProID, but in the case of a number of proteins with low masses (<15 kD), single or pairs of matching MS/MS spectra with very high probabilities of matching (>95%) derived by ProID were evaluated and included in Table II.
LC-MS/MS Analysis of Whole Mitochondrial Protein Extracts
Gel-based arraying of protein samples can exclude proteins from analysis due to hydrophobicity, size, and pI that physically prevents separation in these media. Gel arraying also prevents analysis of peptides that are not easily eluted from polyacrylamide gel samples. The use of reverse-phase chromatography using C18 media provides a gel-free separation of peptides from complex samples. Coupled with on-line MS/MS analysis, the gradual elution of peptides from reverse-phase C18 with organic solvent can be used as a powerful proteomic identification tool. We have taken this approach with a whole-rice mitochondrial protein extract. The identified peptides are matched back to predicted gene products, and the total number of peptides matched provides increasing confidence that the intact gene product was present in the original sample before digestion. Matching of peptides to predicted gene products was again performed using ProID (Applied Biosystems) using mass accuracy cutoffs of peptide mass ± 0.15 and MS/MS ± 0.05. This analysis matched a set of 170 MS/MS spectra back to a set of 72 predicted rice gene products (TableII). For multipeptide matches to a single gene product, a probability cutoff of 85% in ProID was used to filter matches; for single peptide matches, a probability cutoff of 99% in ProID was used for filtering.
A Wide Range of Mitochondrial Functions Identified in Rice
Extensive discussions of many of the components identified here and their biological roles can be found in the earlier reports on the proteomes of Arabidopsis and pea (Kruft et al., 2001;Millar et al., 2001; Bardel et al., 2002). Here, we have largely restricted any extensive comment to components that were not identified in these earlier studies (TableII). The protein sequences for each entry were subjected to BLAST-based similarity analysis to confirm annotation or propose appropriate putative functional annotation for each entry.
Over 30 components of the five electron transport chain complexes from rice were identified. Many of these samples were from BN SDS-PAGE (Fig. 2). This has now allowed the clear identification of the native complexes separating by this technique and noted on the horizontal axis of Figure 2. A significant number of these identifications, although clear orthologs to components of these complexes in other eukaryotic species, have not been reported previously in plant proteome mapping studies. These newly identified components include two subunits of ATP synthase, seven subunits of complex I, and three subunits of complex IV (Table II).
Carbon Metabolism
At least one component of each of the seven tricarboxylic acid cycle enzymes were identified in Table II along with components of the associated pyruvate dehydrogenase complex and the NAD-malic enzyme. The two distinct NAD-malic enzyme subunits have been identified by antibody reaction on IEF/SDS-PAGE from potato (Solanum tuberosum; Jenner et al., 2001) but were not revealed by proteome mapping studies in Arabidopsis or pea. Here, the two subunits were both found on BN SDS-PAGE and appear to be present in separate high-molecular mass complexes. The larger subunit is in a complex with a mass over 300 kD based on its presence between complex III and separated FO of the ATP synthase (Fig.2). The E1 subunit of 2-oxoglutarate dehydrogenase complex was not identified in the Arabidopsis or pea reports, perhaps due to its large mass (Table II). Three components associated with Gly metabolism in mitochondria, the H and T subunits of Gly decarboxylase and the Ser hydroxymethyltransferases, were identified, even though these rice mitochondria were isolated from dark-grown shoots and the photorespiratory requirement for Gly metabolism was absent. A series of 18 enzymes involved in amino acid metabolism (Glu dehydrogenase, Asp transaminase, aldehyde dehydrogenases, and methylmalonate semi-aldehyde dehydrogenase), C1 metabolism (formate dehydrogenase), nucleotide metabolism (inosine-5-monophosphate dehydrogenase and formyl transferase), and miscellaneous metabolic functions were identified. Based on sequence similarity, the formyl transferase is mostly likely a phosphoribosylglycinamide formyl transferase (GART) involved in purine biosynthesis. The potential for plant mitochondria to facilitate nucleotide metabolism has been reported previously (Atkins et al., 1997; Smith et al., 1998). The acetyl-CoA carboxylase is normally involved in long-chain fatty acid synthesis, and although this is commonly considered to be a plastidic function in plants, both mammalian and yeast (Saccharomyces cerevisiae) mitochondria contain this enzyme (Schon, 2001).
Protein and Metabolite Transport
Four components of the import apparatus were identified, two TIM components (subunits 9 and 17) and two from the outer membrane TOM complex (subunits 9 and 40). The latter comigrated during BN electrophoresis (Fig. 2). In Arabidopsis, the TOM complex has to be extensively purified to see its subunits on BN-PAGE (Werhahn and Braun, 2002). Interestingly, the TOM9 and TOM40 subunits represent the core of the TOM complex in yeast, to which other subunits are peripherally attached. A series of specific gene products for the outer membrane voltage-dependent anion channels (porins) were identified, along with three members of the carrier family on the inner mitochondrial membrane responsible for the exchange of inorganic phosphate, ATP/ADP, and dicarboxylates. The Arabidopsis ortholog of the dicarboxylate carrier has been shown recently to be a broad dicarboxylate/tricarboxylate carrier capable of transporting many of the tricarboxylic acid cycle intermediates that enter and exit plant mitochondria (Picault et al., 2002). These carriers were identified mainly through LC-MS/MS, which is in agreement with the difficulty of identifying these proteins in two-dimensional gel separations from Arabidopsis (Millar and Heazlewood, 2003). None of these carrier proteins were identified in the IEF/SDS-PAGE approaches in Arabidopsis or pea (Millar et al., 2001; Bardel et al., 2002).
The mitochondrial-specific superoxide dismutase and the redox carriers, peroxiredoxin and adrenodoxin, identified in Arabidopsis mitochondrial preparations, were also identified here in rice. Members of the major mitochondrial molecular chaperone classes (70, 60, and 10) and the prohibitins were also found. A series of transcription/translation-related proteins were identified, including a translation elongation factor, a Gly-rich RNA-binding protein, and a set of RNA helicases, like those observed in Arabidopsis (Kruft et al., 2001; Millar et al., 2001). In addition, two members of the PPR (pentatricopeptide repeat) family have been found, along with members of the CH2H2 zinc finger and CREB-binding families putatively involved in protein-RNA interactions. Proteins from the PPR class have been implicated in nuclear restoration of cytoplasmic male sterility in plants (Bentolila et al., 2002) and belong to a large family of proteins that may be responsible for the control of RNA stability and editing in plant mitochondria (Small and Peeters, 2000). To our knowledge, this is the first time these proteins have been definitively identified in plant mitochondrial isolates. A series of miscellaneous function proteins include homologs of the ferripyochelin-binding proteins that were also identified in Arabidopsis (Millar et al., 2001). Homologs of the known mitochondrial acid glycoprotein in yeast, Mam33p (Seytter et al., 1998), the coiled-coil mitofilin motor protein for cytoskeleton attachment to mitochondria (Odgren et al., 1996), along with actin itself, were also identified. A set of 23 unknown function proteins have been found for which no clear similarity based on sequence can be proposed, but a very significant number contain predicted mitochondrial-targeting presequences (Table II).
Comparison of the Three Proteome Datasets
Each separation technique identified a series of proteins contributing to a total of 136 nonredundant rice proteins (Table II). Within the total set, each separation technique matched 38% to 53% of the identified proteins. Using the predicted protein sequences, a series of parameters were calculated that included physical characteristics such as molecular mass, pI, and hydrophobicity (GRAVY), and predicted intercellular targeting based on N-terminal sequence as determined by Predotar, TargetP, and MitoProtII. These data were then used for further analysis of the proteome set as outlined below.
More Hydrophobic, Large, and Basic Proteins Were Identified by BN SDS-PAGE and LC-MS Than by IEF/SDS-PAGE Separations
When the protein identifications made by each technique are considered in the context of physical properties of the predicted proteins, such as hydrophobicity, molecular mass, and pI, an interesting pattern emerges (Fig. 3, A–C). A GRAVY score is a single-value indication of the overall hydropathicity of a protein sequence based on the Kyte and Doolittle algorithms; the more positive the score, the more hydrophobic the overall sequence (Kyte and Doolittle, 1982). The addition of thiourea as a chaotropic agent, the advent of new nonionic sulfobetaine detergents, and the use of the non-charged reductant, tributylphosphine, have all been credited with allowing more hydrophobic proteins to enter two-dimensional gels for separation and analysis (Chevallet et al., 1998; Rabilloud, 1998). To date, proteins with GRAVY scores of approximately +0.25 to +0.30 are considered to represent the upper limit of these two-dimensional gel solubility improvements (Herbert, 1999). Our analysis has revealed that the highest GRAVY score for a rice mitochondrial protein identified from IEF/SDS-PAGE was +0.14 for the ATP synthase delta subunit. In BN SDS-PAGE, GRAVY scores reached +0.84 in the case of subunit 9 of the ATP synthase. Overall, there was little difference in the proportion of identifications achieved by the three separation techniques at GRAVY < +0.1, but above this level, more than 60% of the identifications were achieved from BN SDS-PAGE (Fig. 3A).
Comparison of identified rice protein sets. Proportion of rice proteins identified by the three separation techniques (IEF/SDS-PAGE, clear; BN SDS-PAGE, gray; and LC-MS, black) in different classes of hydrophobicity (A), molecular mass (B), and pI (C). D, Overlapping of targeting prediction of the rice proteins identified by TargetP, Predotar, and MitoProtII. Numbers in each circle indicate numbers predicted either by one, two, or all three predictors.
Proteins with large molecular masses can be separated by single-dimension SDS-PAGE. Proteins as large as 200 to 300 kD can be routinely separated using gradient gels. However, such large proteins are not very soluble during IEF and as a result, large proteins (>100 kD) are rarely seen on IEF/SDS-PAGE gels. The proportion of identifications of proteins greater than 80 kD was very significantly shifted toward the LC-MS/MS separations, where size of the parent protein was not an impediment to separation and analysis (Fig. 3B). The basicity of proteins has often restricted identification from IEF/SDS-PAGE due to the difficulty of preparing stable gradients in excess of pH 10 for separation of such proteins. Clearly, in our analysis, it is both the non-IEF gel-separating technique of BN SDS-PAGE and the non-gel LC MS/MS analysis that dominate the identifications of proteins with pI values greater than pH 9 (Fig.3C).
These analyses clearly indicate that to fully characterize mitochondrial proteomes, a series of experimental approaches will be required to maximize the representation of all proteins without bias based on physical characteristics of size, charge, and hydropathicity. LC MS/MS does not appear to be the complete answer, although when many biases are eliminated, a great deal of information about the parent protein from which peptides are derived is lost. Furthermore this technique appears to selectively not identify some proteins present in two-dimensional gel separation of the same material (Table II). Using different gel-based approaches, information on possible posttranslational modifications (such as the multiple spots for malate dehydrogenase, T protein of GDC, alpha-subunit of ATP synthase, and E1β-subunit of pyruvate dehydrogenase subunit; Fig. 1; Table II) and protein-protein interactions (such as components in the electron transport chain and import translocase complexes; Fig. 2; Table II) can be uncovered.
Targeting Prediction of the Rice Proteome Set
The predicted protein sequences of the identified rice proteome were analyzed by three targeting prediction programs commonly used to assess mitochondrial localization in plants (Table II). Five entries in Table II were matched only to rice TCs. It is unknown whether these represent full or partial sequences; thus, they have been excluded from the analysis. Each prediction program assessed that approximately 50 to 60 of the protein products contained N-terminal extensions for mitochondrial targeting. In total, 77 of the 130 protein sequences were predicted to be mitochondrial by at least one targeting program. Looking at the overlapping predictions (Fig. 3D), TargetP identified the largest number of proteins, and the combination of TargetP and Predotar provided the best pair for prediction in rice. In the list of 39 proteins not assessed to be mitochondrial targeted by any program, a large number (19) are well-known mitochondrial proteins involved in the electron transport chain, tricarboxylic acid cycle, import apparatus, and mitochondrial transport. This lack of prediction may be explained either by the absence of targeting presequences (as has been documented for carrier and import components; Millar and Heazlewood, 2003), cryptic presequences not recognized by these program, and/or the lack of presequences due to incorrect annotation of the exons making up N-terminal sequences of these predicted proteins (Frishman et al., 1999). A further three proteins are products of mitochondrial-encoded genes (α-subunit ATP synthase, COXI, and NAD9); thus, they do not require targeting presequences. A total of 11 of the unknown function proteins identified were predicted as mitochondrial targeted by at least one prediction program.
Comparison of Rice, Arabidopsis, and Pea Mitochondrial Proteomes
A comparison between the proteins found here in rice mitochondria and those previously discovered in Arabidopsis (Kruft et al., 2001; Millar et al., 2001) and pea mitochondria (Bardel et al., 2002) is presented in the final two columns of Table II. An asterisk in the second-to-last column indicates that an ortholog was identified in Arabidopsis among the 91 nonredundant set defined by the two Arabidopsis proteome reports and several recent publications containing small sets of proteins. Overall, this analysis indicates that 71 proteins have been found in the mitochondrial proteomes of both species, including two of unknown function. A further set of 28 of the identified rice proteins represent orthologs of well-known mitochondrial proteins in other eukaryotic systems but have not be identified in the published Arabidopsis proteome to date. An asterisk in the last column indicates that an ortholog was found in the nonredundant set of 48 proteins found to date in pea mitochondria. A total of 28 proteins were found in both rice and pea mitochondria. Six of these proteins were not observed in Arabidopsis mitochondria. This leaves a set of 35 proteins, of which 21 have no predicted function, that have been exclusively identified in rice mitochondria to date. This demonstrates the necessity to compare mitochondrial proteomes from different plant species, and most certainly between the major lineages of higher plants, to better understand the complex role of these organelles in the plant cell. However, until a much more complete survey of the proteomes of mitochondria in several plants is conducted in similar tissue types using more similar protein arraying and identification technology, it will be difficult to truly discuss the presence and/or absence of specific proteins between plant species with any accuracy. Currently, many absences in the published Arabidopsis and pea sets reflect technical issues rather than biological absence. Here, we highlight the rice mitochondrial protein complement and present the tools that will be required for such comparative plant proteome investigations in the future.
Growth of Rice Seedlings
Batches of 200 g of rice (Oryza sativa cv Amaroo) seed were washed in 1% (w/v) bleach for 10 min, rinsed in distilled water, and grown in the dark in vermiculite trays (30 × 40cm) at a constant 30°C, watered daily, and shoot tissue harvested at 7 d for mitochondrial isolation.
Rice Mitochondrial Isolation
Rice shoots were harvested using scissors and cut into 5- to 10-mm lengths into a beaker in ice. Cut shoots (100 g) were ground in a precooled mortar and pestle using acid-washed sand in 300 mL of homogenization solution (0.3 m Suc, 25 mmtetrasodiumpyrophosphate, 2 mm EDTA, 10 mmKH2PO4, 1% [w/v] polyvinylpyrrolidone-40, 1% [w/v] bovine serum albumin, and 20 mm ascorbate [pH 7.5]). After filtering homogenate through four layers of Miracloth (Calbiochem, San Diego), it was centrifuged for 5 min at 1,000g, and the supernatant was centrifuged again at 20,000g for 20 min. The resultant organelle pellet was resuspended in wash buffer (0.3 m Suc, 10 mm TES-KOH [pH 7.5], and 0.1% [w/v] bovine serum albumin), and the two centrifugations steps were repeated to produce a washed organelle pellet. This resuspended pellet was layered over 0% to 4.4% (w/v) polyvinylpyrrolidone-40 preformed gradient in a 28% (v/v) Percoll self-forming gradient in wash buffer and centrifuged at 30,000g for 45 min. Mitochondria formed a band toward the bottom of the gradient, the upper plastid material was discarded, and the mitochondrial band was removed in an attempt to minimize contamination of peroxisomal material from the bottom of the gradient. After two wash centrifugation steps at 20,000g for 15 min, the pellet was layered over a second self-forming gradient consisting of 45% (v/v) Percoll in wash buffer and centrifuged for 30 min at 30,000g. Mitochondria remained near the top of the gradient, whereas peroxisomal material migrated to the bottom of the gradient. The mitochondrial layer was aspirated and concentrated by wash centrifugation steps at 20,000g for 15 min.
Respiratory Measurements
O2 consumption was measured in a Clark-type O2 electrode in 1 mL of reaction medium containing: 0.3m mannitol, 10 mm TES-KOH (pH 7.5), 5 mm KH2PO4, 10 mm NaCl, 2 mm MgSO4 and 0.1% (w/v) bovine serum albumin. Pyruvate (5 mm), malate (0.5 mm), succinate (10 mm), NADH (1 mm), ADP (0.5 mm), and myxothiazol (5 μm) were added as indicated to modulate O2 consumption rates. Cytochrome c oxidase activity was measured as ascorbate (5 mm) and cytochrome c (25 μm)-dependent O2 consumption in the presence of 0.05% (w/v) Triton X-100. Outer membrane integrity was assayed as the latency of cytochrome c oxidase activity (Neuberger, 1985).
Two-Dimensional Gel Electrophoresis
Mitochondria protein samples (500 μg) were acetone extracted by addition of acetone to a final concentration of 80% (v/v) at −80°C. Samples were stored at −20°C for 4 h and then centrifuged at 20,000g for 15 min. The pellets were resuspended in an IEF sample buffer consisting of: 6 murea, 2 m thiourea, 2% (w/v) CHAPS, 2% (v/v) ampholytes (pH 3–10), 2 mm tributylphosphine, and 0.001% (w/v) bromphenol blue. Aliquots of 360 μL were used to reswell dried 180-mm, nonlinear immobilized pH gradient strips (pH 3–10; Immobiline DryStrips, APBiotech, Sydney) overnight, and then IEF was performed for 19.5 h, reaching a total of 49 kV h−1 at 20°C on a flat-bed electrophoresis unit (Multiphor II, APBiotech). Immobilized pH gradient strips were then transferred to an equilibration buffer consisting of 50 mm Tris-HCl (pH 6.8), 4 m urea, 2% (w/v) SDS, 0.001% (w/v) Bromphenol blue, and 100 mm mercaptoethanol and incubated for 20 min with rocking. The equilibrated strips were then slotted into central single wells of 4% (w/v) acrylamide stacking gels above 0.1- × 18.5- × 20-cm, 12% (w/v) acrylamide, 0.1% (w/v) SDS-polyacrylamide gels. Strips were overlaid with 0.5% (w/v) agarose in SDS-PAGE running buffer. Gel electrophoresis was performed at 25 mA per gel with circulating cooling and completed in 5 h. Proteins were visualized by colloidal Coomassie (G250) staining. M rand pI standards from APBiotech were used to confirm fixed pH gradient positioning on first dimension separation and to identify apparent molecular masses on second dimension separation.
BN PAGE/SDS-PAGE
BN PAGE was performed largely according to Jansch et al. (1996). Gels consisted of a separating gel (5%–15% [w/v] acrylamide) and a stacking gel (4% [w/v] acrylamide) were formed in a solution of 0.25 mε-amino-n-caproic acid and 25 mmBis-Tris-HCl (pH 7.0). The anode buffer consisted of 50 mmTricine, 15 mm Bis-Tris-HCl, 0.02% (w/v) Blue G250 (pH 7.0), and the cathode buffer of 50 mm Bis-Tris-HCl (pH 7.0). Aliquots of 1 mg of membrane protein were suspended in 75 μL of an amino caproic acid buffer solution (containing 0.75 mε-amino-n-caproic acid, 0.5 mmNa2EDTA, and 50 mm Bis-Tris-HCl [pH 7.0]) followed by the addition of 15 μL of a freshly prepared solution of 10% (w/v) n-dodecylmaltoside. Afterward, 10-min centrifugation at 20,000g was performed. Fifteen microliters of 5% (w/v) Blue G250 was added to the supernatants (dissolved in amino caproic acid buffer). Gels were run at 4°C in a precooled apparatus with all samples, and buffers were precooled to 4°C. Approximately 30 min before the run, 0.03% (w/v)n-dodecylmaltoside was added to the cathode buffer only. Electrophoresis was commenced at 100-V constant voltage for 45 min and then increased to 15 mA without voltage limitation for 5 h. BN lanes were cut from gels, equilibrated in standard SDS-PAGE sample buffer for 30 min, laid horizontally on 12% (w/v) acrylamide SDS-PAGE separating gels, sealed in with 0.5% (w/v) agarose, and electrophoresis was continued for 5 h under standard conditions.
Trypsin Digestion of Gel Plugs and Whole Mitochondrial Extracts
Protein spots to be analyzed were cut from two-dimensional PAGE gels, destained, dried at 50°C, and digested with trypsin according to Sweetlove et al. (2001). Aliquots of 50 μg of purified rice mitochondrial protein were acetone precipitated, and the protein pellets were air dried. A digestion solution consisting of 100 mm Tris-HCl (pH 8.6) and 50 μg mL−1 trypsin was added to a total volume of 45 μL, and the digestion mixture was incubated for 16 h at 37°C.
Quadrupole TOF MS
Quadrupole TOF MS/MS was performed on an Applied Biosystems Q-STAR Pulsar using an IonSpray source. Samples from gel plugs were directly injected into the electrospray source in 50% (v/v) methanol/0.1% (v/v) formic acid, and selected doubly charged peptides, identified in MS-TOF mode, were fragmented by N2 collision and analyzed by MS/MS using IDA software (Applied Biosystems, Foster City, CA). Samples from digestion of whole extracts were bound onto a microbore HPLC C18 column (Agilent, Palo Alto, CA) and eluted over 6 h with a linear acetonitrile gradient from 2% to 80% (v/v) in water. Mass spectra and collision MS/MS data were analyzed with Analyst QS and BioAnalyst software (Applied Biosystems).
Identification of Database Entry Matches and Sequence Analysis
TIGR draft rice genome and rice EST gene indices and rice National Center for Biotechnology Information entries were used for sequence matches. Predotar (http://www.inra.fr/Internet/Produits/Predotar/), TargetP (http://www.cbs.dtu.dk/services/TargetP/), and MitoProtII (http://www.mips.biochem.mpg.de/cgi-bin/proj/medgen/mitofilter) predictions were determined using full-length predicted protein sequences. Molecular mass, pI, and GRAVY scores were determined using the ProtParam program on an ExPASy Web site (http://au.expasy.org).
↵1 This work was supported by the Australian Research Council Discovery Program (to A.H.M. and J.W.). K.A.H. was a recipient of the Eric Cyril Lawrence Medical Research Scholarship.
↵* Corresponding author; e-mail hmillar{at}cyllene.uwa.edu.au; fax 61–8–9380–7245.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.102.018986.
Revision received January 10, 2003.
Accepted February 3, 2003.
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You are going to email the following Towards an Analysis of the Rice Mitochondrial Proteome
Plant Physiology May 2003, 132 (1) 230-242; DOI: 10.1104/pp.102.018986
Long-Term Acclimation of the Cyanobacterium Synechocystis sp. PCC 6803 to High Light Is Accompanied by an Enhanced Production of Chlorophyll That Is Preferentially Channeled to Trimeric Photosystem I
Truncated Photosystem Chlorophyll Antenna Size in the Green Microalga Chlamydomonas reinhardtii upon Deletion of the TLA3-CpSRP43 Gene
Steady-State Phosphorylation of Light-Harvesting Complex II Proteins Preserves Photosystem I under Fluctuating White Light
Show more BIOENERGETICS AND PHOTOSYNTHESIS


text:
Главная / News / Mining industry / KZhRK miners' strike threatens with loss of sales markets without prospects of their return
KZhRK miners' strike threatens with loss of sales markets without prospects of their return
Mining industry / Ukraine
This statement was made in the press service of PJSC "Kryvyi Rih Iron Ore Plant" against the background of the ongoing protest of miners dissatisfied with their wages.
The stoppage of production has already caused appeals from European consumers of the products of the PJSC "Kryvyi Rih Iron Ore Plant" regarding non-compliance with the terms of the concluded contracts, and also threatens to lose markets in favor of other suppliers with a weak prospect of their return. stated in press service of PJSC "Kryvyi Rih Iron Ore Plant" (KZhRK).
On behalf of the administration, in order to resolve the labor conflict, as well as prevent the spread of rumors and falsifications, the press service of the KZHRK decided to inform the employees of the enterprise, heads of trade unions, the country's leadership, residents of Krivoy Rog and citizens of Ukraine about the situation at the enterprise, measures were taken and the state of negotiations with mine workers.
As of 8:00 on September 16, a group of miners from the Rodina (76), Oktyabrskaya (25), Gvardeyskaya (34) and Ternovskaya (49) mines, 184 people on their own the initiative and in violation of the work schedule remain in the underground workings.
Among the miners' demands are an increase in wages, dismissal of management, return of preferential service according to lists No. 1 and No. 2 and suppression of violations during certification.
The press service of KZHRK focused on the fact that due to the fact that written requirements from the labor collective did not come to the administration of the enterprise and, accordingly, the administration did not declare refusals to fulfill any written requirements of the labor collective, in accordance with the current by law, the actions of the protesters do not fall under the definition of a strike and are not a strike.
"In addition, the law expressly stipulates (Article 24, first part) that it is prohibited to hold a strike, provided that the termination of work by workers poses a threat to the life and health of people, the environment or prevents a natural disaster, accidents, catastrophes," they noted in the press service of KZhRK.
They recalled that the administration of the enterprise, on its own initiative, adopted and promulgated the "Memorandum of Understanding and the fulfillment of counter-requirements between the administration and employees of PJSC" Kryvbaszhelezrudkom "aimed at eliminating the causes and consequences, due to the shifts ". In the document, the administration unilaterally announced its readiness to resolve the conflict.
According to the Memorandum, order No. 3850 of 09/10/2020 "On the increase in tariff rates and official salaries from 09/01/2020" from 10% to 25% was issued. According to order No. 3864 of 09/11/2020, the list of occupations of workers for whom tariff rates and official salaries are increased by 25% have been supplemented.
For the purpose of further settlement, the administration of PJSC "Kryvyi Rih Iron Ore Plant" sent an appeal to the chairman of the primary trade union organization of the Independent Trade Union of Miners of Ukraine, PJSC "Kryvyi Rih Iron Ore Plant" and to the chairman of the primary organization of the Trade Union of Workers of the Metallurgical and Mining Industry of Ukraine, PJSC "Kryvyi Rih Iron Ore Plant" with a proposal to create a joint commission to review pay systems.
In recent days, the administration of PJSC "Kryvyi Rih Iron Ore Plant" has repeatedly appeared in open sources and continues to insist on establishing a constructive dialogue with all interested parties in order to resolve the conflict by looking for a compromise based on the requirements of current legislation.
KZhRK emphasized that the further stay of the protesters in the underground workings without actual intention to resolve the conflict through a constructive dialogue with the administration in the manner determined by Ukrainian legislation not only endangers the employees of the enterprise, but also creates consequences that will negatively affect work for a long period. reduce the ability of the enterprise to fulfill its financial responsibilities.
The press service of the KZHRK warned politicians against attempts to use the current situation at the enterprise to achieve personal political goals, inciting employees of the enterprise and their families to illegal and destructive actions.
"All responsibility for the possible deterioration of the health of persons who are in the underground workings in protest, as well as the inevitable, probably irreversible negative consequences for the enterprise, fall on such irresponsible political adventurers. Once again we emphasize the desire of the administration to move in a constructive direction, in accordance with the current legislation of Ukraine, to resolve the conflict situation and restore the operation of the enterprise, "the press service of KZHRK summed up.
Руководитель КЖРК распорядился о подготовке к консервации всех четырех шахт предприятия – Волынец
ПАО «Кривбассжелезрудком» намерено принудительно поднять шахтеров из шахты
Ukraine accepted the offer of the Mask on the free ventilators
Belanto Trade accused Dmitry Firtash of tarnishing the company's image to eliminate a competitor
Metinvest is recognized as one of the most sustainable companies in Ukraine
Uz with EBRD assistance will repair the TRANS-European transport corridor
Ukrainian-made gas equipment for the export of hydrogen to Europe is being tested in Ivano-Frankivsk
Rinat Akhmetov's engineering company Corum Group sent a delegation to India
Zaporizhstal Metallurgical Plant mastered eight new types of products in 2022
Europe's passenger car market records its worst year since the turn of the century

redpajama_set_name: RedPajamaCommonCrawl


Talk to system retailers, talk to others who already have systems, and do other research to choose the best system and retailer for your needs. Cheaper may not be better, and bigger may not be better for you. Check our Solar PV webpage for more information.
To get your system connected to our network, you need to apply for a new connection contract with us. As technical information is required, it's a good idea to have your system retailer apply for you. Otherwise you, or someone on your behalf, can apply via our Customer Portal.
If you have any questions, you can call us on 13 12 53 (Mon to Fri, 7am - 5:30pm). Otherwise, you can lodge an enquiry online via our Customer Portal. We'll respond within five business days.
When we receive your application, we'll check if the connection service needed for your system is basic or negotiated. We may also check if the local network can support your new system.
We do a technical assessment of most applications classed as negotiated. If our technical assessment finds your proposed system may not work effectively on our network, we will provide recommendations through our Customer Portal to advise you of the options available. Once you, or your system retailer, select an option and update your application, we'll arrange for your connection offer to be sent.
Read more about connection service types, offers and contracts under FAQs and further reading below.
Your system installer or electrical contractor (try Master Electricians) must submit an Electrical Work Request (EWR) as soon as your system has been installed. This is to advise us that the work at your premises has been carried out in accordance with the required standards.
The EWR will also notify your electricity retailer of your new system installation. Your retailer may then contact you to discuss any metering changes required so it can measure any electricity you export to the grid. You will need to discuss feed-in tariff options with your electricity retailer.
Important: Your installer must leave your system switched off and you must not switch it on. Once your meter has been updated to enable the feed-in-tariff, you will be advised you can turn on your system.
For an easy print summary of this connection process, check out our Connect solar PV or batteries checklist (PDF 186.3 kb).
Please note: If you request information by post, it may take longer for you to receive your offer.
To get your system connected, we need an EWR from your installer so we can advise your electricity retailer of your request. Your retailer will advise the timeframe for any metering changes and when you can turn your system on.
All our connection charges have been approved by the Australian Energy Regulator. Read more on connection charges.
There will be fees to install or enable the required electricity metering at your premises. The amount is charged to you by your electricity retailer and you will need to discuss these charges with them.
Your installer or licenced electrical contractor will advise you of any work required to prepare your premises for the installation of your system, for example upgrading your switchboard. We recommend you talk to them for more information on the work needed at your place.
To ensure the safe and reliable connection of your solar PV system with our network, there are a number of things you need to know. Read more in Responsibilities of owning a micro embedded generator (solar PV system) (Form 8007) (PDF 356.1 kb).
The owner of your premises if you are a tenant.
you can also call us on 13 12 53 (Mon to Fri, 7am - 5:30pm) to request a form.
I don't want to use an online form do you have a printed form I can use?
Yes, we have printed versions of our connection enquiry form and application form that we can send to you.
You can call us on 13 12 53 (Monday to Friday 7:00am - 5:30pm) to request a form.
An embedded generating unit is connected directly within an electricity distribution network and does not have direct access to the electricity transmission network.
To be a micro embedded generating unit, it must be fitted with an inverter, such as in a solar PV system (this excludes rotating machines), and be compliant with the technical requirements of AS/NZS 4777. Previously, AS/NZS 4777 only included generators up to 30 kVA. However, it has recently been changed to cover embedded generators up to 200 kVA.
If you are interested in connecting a generating system larger than 30 kVA, please refer to our Large Generation webpage.
One of our technical advisors will advise you of any changes required to your application.
Eligibility for the regional feed-in tariff under the Queensland Government's Solar Bonus Scheme depends on the total capacity of your system, your electricity retailer and other criteria. More details are available on our Solar Bonus Scheme webpage.
Read more on how to apply to connect a larger scale embedded generating system (like solar, wind, hydro or diesel) above 30 kVA on our Large Generation webpage.
Systems that qualify for a basic connection service are typically exporting inverters rated up to 5 kVA.
Our standard connection services apply to unmetered supply only, for example watchman lighting, bus shelters, lighting for public amenities or street lighting. The standard connections services we offer are listed in our Model Standing Offer - Standard Connection Service for Unmetered Supply (PDF 343.1 kb).
We'll then make a basic or negotiated connection offer within set timeframes for you to accept.
The basic connection services that we offer are listed in our Model Standing Offer Basic Connection Service (MEG) (PDF 425.2 kb).
For basic connection service, these terms are listed in our Model Standing Offer Basic Connection Service (MEG) (PDF 425.2 kb).
The types of basic connections for micro embedded generating units are outlined in basic connection services.
For negotiated connection services, we'll negotiate the terms of your connection contract with you.
Once you have accepted the connection offer and your connection establishment contract is in place, another contract automatically comes into effect for your ongoing connection services. Unless you specifically negotiate the terms of this other contract, it will be on the terms of the Deemed Standard Connection Contract (PDF 704.5 kb).
A service line is the wire that runs from our network to just inside your property and connects electricity to your premises. A standard overhead service line may extend approximately 20m inside your premises, whilst a standard underground service line may extend approximately 7m inside your premises.
A capital contribution for other network changes may also apply if the new connection or connection alteration exceeds the network change charge threshold. The threshold is currently set at a maximum capacity of 100 amperes on 3 phase low voltage supply.
In these circumstances, any capital contributions will be calculated using the formula set out in section 7 in our Connection Policy (PDF 1.1 mb).
Certain subdivision works (visit Residential subdivisions or Business subdivisions).
Certain large customer connection works (visit Major business connections).


text:
Q: MS Access VBA temporarily highlight field When a user switches between records on a sub form, I want another field to highlight temporarily to catch the eye to alert the user that it has changed.
I would think that this would work:
Declare Sub Sleep Lib "kernel32" (ByVal dwMilliseconds As Long)

Private Sub Form_Current()
    Form_frm_Codes.txtCodeToAdd = Me.Code.Value
    Form_frm_Codes.txtCodeToAdd.BackColor = RGB(0, 0, 255)
    Sleep (500)
    Form_frm_Codes.txtCodeToAdd.BackColor = RGB(255, 255, 255)
End Sub

The sleep function tends to pause the program for half a second, but I don't see the blue change imediately before.
Any ideas or even a better way to achieve this?

A: Instead of using API try below code
Private Sub Form_Current()
    Form_frm_Codes.txtCodeToAdd = Me.Code.Value
    Form_frm_Codes.txtCodeToAdd.BackColor = RGB(0, 0, 255)
    delay 5
    Form_frm_Codes.txtCodeToAdd.BackColor = RGB(255, 255, 255)
End Sub

Private Sub delay(seconds As Long)
    Dim endTime As Date
    endTime = DateAdd("s", seconds, Now())
    Do While Now() < endTime
        DoEvents
    Loop
End Sub

OR add DoEvents before sleep to update the screen.
Private Sub Form_Current()
    Form_frm_Codes.txtCodeToAdd = Me.Code.Value
    Form_frm_Codes.txtCodeToAdd.BackColor = RGB(0, 0, 255)
    DoEvents
    Sleep (500)
    Form_frm_Codes.txtCodeToAdd.BackColor = RGB(255, 255, 255)
End Sub

redpajama_set_name: RedPajamaStackExchange


text:
Q: Setting up a if attribute For Example > if content = tree return special content to tree
content being > index.php?content=tree
<?php
if (<?= $_GET['content'] ?> == tree) {
echo "text here";
}
else {
echo "nothing to see here";
}
?>

A: Fixed up:
<?php
if (isset($_GET['content']) && $_GET['content'] == 'tree') {
echo "text here";
}
else {
echo "nothing to see here";
}
?>

Multiple:
<?php
if (isset($_GET['content']) && in_array($_GET['content'], array('tree', 'bird', 'fish', 'taco')) ) {
echo "text here";
}
else {
echo "nothing to see here";
}
?>

A: First i advice you check functions and other php code on the php manual, Heres an example of the if/elseif/else statement: link
Now in your code you did this:     if (<?= $_GET['content'] ?> == tree) {
Note how you called php echo     <?= $_GET['content'] ?> == tree as a condition, first mistake there since you cant call php as a condition, not to mention echo.
this is how you should have wrote the code:
<?php

    if ($_GET['content'] == 'tree') {

        echo "text here";

    }
    else {

        echo "nothing to see here";

    }

?>

Please check out the link i gave you.


text:
Q: How to store spark streaming data to Hdfs in Hortonworks? I have streamed data from Kafka topics using Spark. This is the code that I have tried. Here I was just displaying the streaming data in console. I want to store this data as a text file in HDFS.
import _root_.kafka.serializer.DefaultDecoder
import _root_.kafka.serializer.StringDecoder
import org.apache.spark.SparkConf
import org.apache.spark.streaming.{Seconds, StreamingContext}
import org.apache.spark.streaming.kafka.KafkaUtils
import org.apache.spark.storage.StorageLevel
object StreamingDataNew {
  def main(args: Array[String]): Unit = {
    val sparkConf = new SparkConf().setAppName("Kafka").setMaster("local[*]")
    val ssc = new StreamingContext(sparkConf, Seconds(10))
val kafkaConf = Map(
      "metadata.broker.list" -> "localhost:9092",
      "zookeeper.connect" -> "localhost:2181",
      "group.id" -> "kafka-streaming-example",
      "zookeeper.connection.timeout.ms" -> "200000"
    )
val lines = KafkaUtils.createStream[Array[Byte], String, DefaultDecoder, StringDecoder](
      ssc,
      kafkaConf,
      Map("topic-one" -> 1), // subscripe to topic and partition 1
      StorageLevel.MEMORY_ONLY
    )
    println("printing" + lines.toString())
    val words = lines.flatMap { case (x, y) => y.split(" ") }
    words.print()

    ssc.start()
    ssc.awaitTermination()

  }
}

I found that we can write the DStream using 'saveAsTextFiles'.But can someone clearly mention the steps on how to connect with Hortonworks and store in HDFS using the above scala code.

A: I found the answer,
This code worked out for me.
package com.spark.streaming

import org.apache.kafka.common.serialization.StringDeserializer
import org.apache.spark.SparkContext
import org.apache.spark.sql._
import org.apache.spark.streaming.StreamingContext
import org.apache.spark.streaming.kafka010.ConsumerStrategies.Subscribe
import org.apache.spark.streaming.kafka010.KafkaUtils
import org.apache.spark.streaming.kafka010.LocationStrategies.PreferConsistent

object MessageStreaming {
  def main(args: Array[String]): Unit = {
    println("Message streaming")

    val conf = new org.apache.spark.SparkConf().setMaster("local[*]").setAppName("kafka-streaming")
    val context = new SparkContext(conf)
    val ssc = new StreamingContext(context, org.apache.spark.streaming.Seconds(10))
    val kafkaParams = Map(
      "bootstrap.servers" -> "kafka.kafka-cluster.com:9092",
      "group.id" -> "kafka-streaming-example",
      "key.deserializer" -> classOf[StringDeserializer],
      "value.deserializer" -> classOf[StringDeserializer],
      "auto.offset.reset" -> "latest",
      "zookeeper.connection.timeout.ms" -> "200000"
    )
    val topics = Array("cdc-classic")
    val stream = KafkaUtils.createDirectStream[String, String](
      ssc,
      PreferConsistent,
      Subscribe[String, String](topics, kafkaParams))

    val content = stream.filter(x => x.value() != null)
    val sqlContext = new org.apache.spark.sql.SQLContext(context)
    import sqlContext.implicits._

    stream.map(_.value).foreachRDD(rdd => {

      rdd.foreach(println)
      if (!rdd.isEmpty()) {
     rdd.toDF("value").coalesce(1).write.mode(SaveMode.Append).json("hdfs://dev1a/user/hg5tv0/hadoop/MessagesFromKafka")

      }

    })
    ssc.start()
    ssc.awaitTermination

}}


text:
Grigorij Petrovics Danyilevszkij (oroszul: Григо́рий Петро́вич Даниле́вский, ukránul: Григорій Петрович Данилевський; Danilovka (harjkovi kormányzóság), 1829. április 14. (ó-naptár) – Szentpétervár, 1890. december 6.) orosz regényíró.

Élete

A szentpétervári egyetemen szerezte diplomáját. Tanulmányai végeztével a közoktatásügyi minisztériumban szolgált, utazásokat tett, könyvházakban búvárkodott, majd visszament atyai birtokára és később Pétervárra, ahol a Pravitjelsztvennyj Vjesztnik című hivatalos lapot alapították meg az ő szerkesztésével 1869-ben. Ezt a lapot szerkesztette egész haláláig. Írói pályáját már 1846-ban megkezdte egy verssel, Byron modorában, később az elbeszélésre adta magát és ezen a téren volt ő igazán otthon az ő karakterisztikus beszélyeivel, regényeivel és korrajzaival, melyekben az orosz életet ritka szerencsés kézzel festette, úgyhogy műveivel csakhamar külföldön is hírnevet szerzett. Legsikerültebb regényei: Bjeglyje o Novorosszii (Menekülők Újoroszországban, 1864, magyarra fordította Szabó Endre Uj Oroszország cím alatt a Nemzet tárcájában); Bjeglyje vorotjilisz (A menekülők visszatérése, 1865); Valja (1865) Szabadság (magyarra fordította Szabó Endre a Budapesti Ujság tárcájában); Devjatyj Val (1874, magyarra fordította Kilencedik hullám cím alatt Szabó Endre a Pesti Hírlap tárcájában); Mirovics (1879); Csjornyj god (Fekete év, történelmi regény Pugacsev korából, 1888); ezen kívül számtalan apróbb elbeszélés, rajz és cikk. Összes művei nyolc nagy kötetben jelentek meg Szentpétervárott 1888-ban.

Magyarul

Danilevszky Péterː Tarakanov hercegnő. Történeti regény; ford. Szalai Emil; Magyar Irodalmi Rt., Bp., 189? (A Magyar Újság regénycsarnoka)

Források

Danilevskii, Grigorii Petrovich The Free Dictionary

Orosz írók
1829-ben született személyek
1890-ben elhunyt személyek

redpajama_set_name: RedPajamaWikipedia


Why not write a brief review about how well Kinectimals ran on your hardware?
Kinectimals is a video game for the Xbox 360 that use Kinect and also available for these platforms: Windows Phone, iOS and Android. In Kinectimals players can interact with virtual animals in a manner akin to pets, gameplay includes activities such as teaching tricks to the animal, guiding the animal around an obstacle course, and free-form play.The video game is aimed at a young audience, particularly children, and includes eleven different virtual animals all based on wild cats. Kinectimals was developed by Frontier Developments and was a launch title for Kinect.An add-on, Kinectimals: Now With Bears!, was released on October 11, 2011.
Please login to add your score for Kinectimals Graphics played on the xbox360.
Please login to add your score for Kinectimals Lifespan played on the xbox360.
Please login to add your score for Kinectimals Value played on the xbox360.


In 2011, I was introduced to the world of internet marketing when I started to promote our direct selling products.
From that day on, I learned the ins and outs of internet marketing. However, as a newbie, there are some frustrations that I encounter the same as yours.
I was not scammed, because I am careful in selecting programs. But, I would like to tell you that by joining these programs I learned a lot.
Every now and then I subscribe to countless emails, newsletter to get those freebies just like what you are doing when you get this one.
But my sad realization is that, ONLINE MARKETING is just a "cycle of making money from innocent newbies".
I realized that I was not doing what I should do, instead I was a part of those who are no direction.
There are many good programs out there and a lot of marketing tools that are promoted.
But in the end, who are making money?
It is the product creator.
But there are really programs that are purely a milking cow for product creators. They will tell you why you need such product or program, but they won't tell you how to start your own online empire.
In the end, you end up buying all of these softwares and programs without earning yourself money. I would not discredit the creators because in the end, it also depends on how you use it.
It's true that your passions will elevate you and your business. If you love cooking, you can make a blog and teach others how to cook and earn from it.
When I was exposed to this online marketing, I did try almost everything to make money from it. In fact, I had build two sites but those are closed.
There was even an incident where I earned money accidentally from posting a link in one of my free blog account. But these are not so successful.
Why I do not succeed in these things?
This brings me to my second tip.
Just this May 2015 I was able to build my third site. Even though this is my third site, I still decided not to focus on making money online as a niche, instead I focused on educating my readers and followers about my advocacy.
Money is just a principle to live by. It is how to appreciate the things that we have.
Many of advertising company allure you with such promises to get rich but is not true in the end.
Focus on course of action until successful!
Many of the newbies are eager to make some money. But just like offline business, it takes years to build it.
Don't be fooled by such push button riches.
There are no failures, only results.
So, keep doing and testing until you find your niche – your customer base.
There are many ways to make money online, but what I want you to realize is that, you have to keep FOCUS and do what you love.
If you are doing affiliate marketing, then continue doing it. Find ways to be successful in those field, but if until now you are struggling to make money online, STOP.
Analyse what's missing in your business.
Do you promote the right product at the right people?
These things are important in online marketing.
Some say, adsense is good way to earn money.
Some would also suggest pay per click is an effective advertising.
But it depends on how you use it.
Don't go after the money. Help people make money and you will earn money.
So, again and again stay FOCUS.
If you are a frustrated online seller or marketer, you have to strategize. Take note, not all emails that you send will be read. So, you must be initiative in crafting those email headlines.
My advice is just don't send them promotional emails. Instead, PRESELL. You must educate them. Then, through it, you win their confidence and you know whats next.
Sending emails with headlines are good, but it's important that you have to have this genuine and sincere goal to help your customers.
Know their needs and aspirations.
If you make them special. Give them value. This way you will there hearts. And whatever you promote or ask to them, it will be granted.
It's because they trust you. They have confidence in you.
Unlike if you are just automailing them with those crappy spammy headlines.
To show you how important you are to me as my customer and reader, I made it as a choice not to include affiliate links to earn money from you in this book.
Because making money is not my mission.
My mission is to help you.
Not to make money from you. If I want to really make money from you, I could have sold this for $ 29.
So, as for you, to genuinely make money online.
Help other people make money.
Don't collect money, if you are not able to give value to your customers.
Bade them to follow you.
This principle is biblical. And it works even in business.
On the other hand, Robert Caildini's principle of influence is this.
Freely give and when you asked them something, they cannot refuse anymore because of gratitude. This is the process of reciprocation.
Remember the first time you receive a free taste of a cake or hamburger? The next time you taste it is not anymore free.
Are you desperate in making money online?
You too can earn money doing what you love.
Here are some of my tips to earn money doing what you always do.
I have been in Fiverr since 2012 but its only this year that I earned over $400 doing what I like. I am a whiteboard video creator and I helped small entrepreneurs make their ideas come true with my special tools for video editing.
What I did is just join Fiverr and post a gig there.
So, don't waste you time.
You can earn money by clicking on those links. But the good thing is that you can earn by refering your friends too.
Payout is $2 for upgraded members. If you have a website or blog, you can advertise your site there for a cheaper cost.
Alternatively, you can join Traffic Monsoon too.
Similar to Clicksense, but with a cheaper traffic pay. Only for $5, you can get 2500 real targeted traffic. It will help you build your own list in almost no time. (Depends on your niche too).
You can get cash links too.
Its one of my favorite, if you like products, whether digital or physical product.
You can have affiliated to the company. If you can promote them, then you will earn money and at the same time doing what you love.
Clickbank is a nice network with lots of products.
Making money online can be frustrating.