ClpB-cytoplasmic (ClpB-cyt)/Hsp100 is an important chaperone protein in rice. Cellular expression of OsClpB-cyt transcript is governed by heat stress, metal stress, and developmental cues. Transgenic rice plants produced with 2 kb OsClpB-cyt promoter driving Gus reporter gene showed heat- and metal-regulated Gus expression in vegetative tissues and constitutive Gus expression in calli, flowering tissues, and embryonal half of seeds. Rice seedlings regenerated with OsClpB-cyt promoter fragment with deletion of its canonical heat shock element sequence (HSE−273 to −280) showed not only heat shock inducibility of Gus transcript/protein but also constitutive expression of Gus in vegetative tissues. It thus emerges that the only classical HSE present in OsClpB-cyt promoter is involved in repressing expression of OsClpB-cyt transcript under unstressed control conditions. Yeast one-hybrid assays suggested that OsHsfA2c specifically interacts with OsClpB-cyt promoter. OsHsfA2c also showed binding with OsClpB-cyt and OsHsfB4b showed binding with OsClpB-cyt; notably, interaction of OsHsfB4b was seen for all three OsClpB/Hsp100 protein isoforms (i.e., ClpB-cytoplasmic, ClpB-mitochondrial, and ClpB-chloroplastic). Furthermore, OsHsfB4b showed interaction with OsHsfA2c. This study suggests that OsHsfA2c may play a role as transcriptional activator and that OsHsfB4b is an important part of this heat shock responsive circuitry.
Electronic supplementary material
The online version of this article (doi:10.1007/s12192-011-0303-5) contains supplementary material, which is available to authorized users.
cis-Acting sequences; Heat shock factor (Hsf); Heat shock protein100 (Hsp100); High temperature; Rice; trans-Acting factors
Cytoplasmic male sterility (CMS) has often been associated with abnormal mitochondrial open reading frames. The mitochondrial gene orfH79 is a candidate gene for causing the CMS trait in CMS-Honglian (CMS-HL) rice. However, whether the orfH79 expression can actually induce CMS in rice remains unclear.
Western blot analysis revealed that the ORFH79 protein is mainly present in mitochondria of CMS-HL rice and is absent in the fertile line. To investigate the function of ORFH79 protein in mitochondria, this gene was fused to a mitochondrial transit peptide sequence and used to transform wild type rice, where its expression induced the gametophytic male sterile phenotype. In addition, excessive accumulation of reactive oxygen species (ROS) in the microspore, a reduced ATP/ADP ratio, decreased mitochondrial membrane potential and a lower respiration rate in the transgenic plants were found to be similar to those in CMS-HL rice. Moreover, retarded growth of primary and lateral roots accompanied by abnormal accumulation of ROS in the root tip was observed in both transgenic rice and CMS-HL rice (YTA).
These results suggest that the expression of orfH79 in mitochondria impairs mitochondrial function, which affects the development of both male gametophytes and the roots of CMS-HL rice.
Reliability of real-time PCR (RT-qPCR) data is dependent on the use of appropriate reference gene(s) for normalization. To date, no validated reference genes have been reported for normalizing gene expression in human myocardium. This study aimed to identify validated reference genes for use in gene expression studies of failed and non-failed human myocardium.
Bioinformatic analysis of published human heart gene expression arrays (195 failed hearts, 16 donor hearts) was used to identify 10 stable and abundant genes for further testing. The expression stability of these genes was investigated in 28 failed and 28 non-failed human myocardium samples by RT-qPCR using geNorm software.
Signal recognition particle 14 kDa (SRP14), tumor protein, translationally-controlled 1 (TPT1) and eukaryotic elongation factor 1A1 (EEF1A1) were ranked the most stable genes. The commonly used reference gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was ranked the least stable of the genes tested. The normalization strategy was tested by comparing RT-qPCR data of both normalized and raw expression levels of brain natriuretic peptide precursor (NPPB), a gene known to be up-regulated in heart failure. Non-normalized levels of NPPB exhibited a marginally significant difference between failed and non-failed samples (p = 0.058). In contrast, normalized NPPB expression levels were significantly higher in heart-failed patients compared with controls (p = 0.023).
This study used publicly available gene array data to identify a strategy for normalization involving two reference genes in combination that may have broad application for accurate and reliable normalization of RT-qPCR data in failed and non-failed human myocardium.
To accurately assess gene expression levels, it is essential to normalize real-time quantitative PCR (RT-qPCR) data with suitable internal reference genes. For the red imported fire ant, Solenopsis invicta, reliable reference genes to assess the transcript expression levels of the target genes have not been previously investigated. In this study, we examined the expression levels of five candidate reference genes (rpl18, ef1-beta, act, GAPDH, and tbp) in different developmental stages, castes and tissues of S. invicta. To evaluate the suitability of these genes as endogenous controls, three software-based approaches (geNorm, BestKeeper and NormFinder) and one web-based comprehensive tool (RefFinder) were used to analyze and rank the tested genes. Furthermore, the optimal number of reference gene(s) was determined by the pairwise variation value. Our data showed that two of the five candidate genes, rpl18 and ef1-beta, were the most suitable reference genes because they have the most stable expression among different developmental stages, castes and tissues in S. invicta. Although widely used as reference gene in other species, in S. invicta the act gene has high variation in expression and was consequently excluded as a reliable reference gene. The two validated reference genes, rpl18 and ef1-beta, can be widely used for quantification of target gene expression with RT-qPCR technology in S. invicta.
High temperature is a critical abiotic stress that reduces crop yield and quality. Rice (Oryza sativa L.) plants remodel their proteomes in response to high temperature stress. Moreover, phosphorylation is the most common form of protein post-translational modification (PTM). However, the differential expression of phosphoproteins induced by heat in rice remains unexplored.
Phosphoprotein in the leaves of rice under heat stress were displayed using two-dimensional electrophoresis (2-DE) and Pro-Q Diamond dye. Differentially expressed phosphoproteins were identified by MALDI-TOF-TOF-MS/MS and confirmed by Western blotting.
Ten heat-phosphoproteins were identified from twelve protein spots, including ribulose bisphos-phate carboxylase large chain, 2-Cys peroxiredoxin BAS1, putative mRNA binding protein, Os01g0791600 protein, OSJNBa0076N16.12 protein, putative H(+)-transporting ATP synthase, ATP synthase subunit beta and three putative uncharacterized proteins. The identification of ATP synthase subunit beta was further validated by Western-blotting. Four phosphorylation site predictors were also used to predict the phosphorylation sites and the specific kinases for these 10 phosphoproteins.
Heat stress induced the dephosphorylation of RuBisCo and the phosphorylation of ATP-β, which decreased the activities of RuBisCo and ATP synthase. The observed dephosphorylation of the mRNA binding protein and 2-Cys peroxiredoxin may be involved in the transduction of heat-stress signaling, but the functional importance of other phosphoproteins, such as H+-ATPase, remains unknown.
Housekeeping genes have been commonly used as reference to normalize gene expression and protein content data because of its presumed constitutive expression. In this paper, we challenge the consensual idea that housekeeping genes are reliable controls for expression studies in the retina through the investigation of a panel of reference genes potentially suitable for analysis of different stages of retinal development.
We applied statistical tools on combinations of retinal developmental stages to assess the most stable internal controls for quantitative RT-PCR (qRT-PCR). The stability of expression of seven putative reference genes (Actb, B2m, Gapdh, Hprt1, Mapk1, Ppia and Rn18s) was analyzed using geNorm, BestKeeper and Normfinder software. In addition, several housekeeping genes were tested as loading controls for Western blot in the same sample panel, using Image J. Overall, for qRT-PCR the combination of Gapdh and Mapk1 showed the highest stability for most experimental sets. Actb was downregulated in more mature stages, while Rn18s and Hprt1 showed the highest variability. We normalized the expression of cyclin D1 using various reference genes and demonstrated that spurious results may result from blind selection of internal controls. For Western blot significant variation could be seen among four putative internal controls (β-actin, cyclophilin b, α-tubulin and lamin A/C), while MAPK1 was stably expressed.
Putative housekeeping genes exhibit significant variation in both mRNA and protein content during retinal development. Our results showed that distinct combinations of internal controls fit for each experimental set in the case of qRT-PCR and that MAPK1 is a reliable loading control for Western blot. The results indicate that biased study outcomes may follow the use of reference genes without prior validation for qRT-PCR and Western blot.
The cbnA gene encoding the chlorocatechol dioxygenase gene from Ralstonia eutropha NH9 was introduced into rice plants. The cbnA gene was expressed in transgenic rice plants under the control of a modified cauliflower mosaic virus 35S promoter. Western blot analysis using anti-CbnA protein indicated that the cbnA gene was expressed in leaf tissue, roots, culms, and seeds. Transgenic rice calluses expressing the cbnA gene converted 3-chlorocatechol to 2-chloromucote efficiently. Growth and morphology of the transgenic rice plants expressing the cbnA gene were not distinguished from those of control rice plants harboring only a Ti binary vector. It is thus possible to breed transgenic plants that degrade chloroaromatic compounds in soil and surface water.
To obtain reliable quantitative real-time PCR data, normalization relative to stable housekeeping genes (HKGs) is required. However, in practice, expression levels of 'typical' housekeeping genes have been found to vary between tissues and under different experimental conditions. To date, validation studies of reference genes in pigs are relatively rare and have never been performed in porcine alveolar macrophages (AMs). In this study, expression stability of putative housekeeping genes were identified in the porcine AMs in response to the stimulation with two pathogen-associated molecular patterns (PAMPs) lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Three different algorithms (geNorm, Normfinder and BestKeeper) were applied to assess the stability of HKGs.
The mRNA expression stability of nine commonly used reference genes (B2M, BLM, GAPDH, HPRT1, PPIA, RPL4, SDHA, TBP and YWHAZ) was determined by qRT-PCR in AMs that were stimulated by LPS and LTA in vitro. mRNA expression levels of all genes were found to be affected by the type of stimulation and duration of the stimulation (P < 0.0001). geNorm software revealed that SDHA, B2M and RPL4 showed a high expression stability in the irrespective to the stimulation group, while SDHA, YWHAZ and RPL4 showed high stability in non-stimulated control group. In all cases, GAPDH showed the least stability in geNorm. NormFinder revealed that SDHA was the most stable gene in all the groups. Moreover, geNorm software suggested that the geometric mean of the three most stable genes would be the suitable combination for accurate normalization of gene expression study.
There was discrepancy in the ranking order of reference genes obtained by different analysing algorithms. In conclusion, the geometric mean of the SDHA, YWHAZ and RPL4 seemed to be the most appropriate combination of HKGs for accurate normalization of gene expression data in porcine AMs without knowing the type of bacterial pathogenic status of the animals.
Candidate reference genes; Alveolar macrophage; LPS; LTA; Pigs
Real-time reverse transcription PCR (RT-qPCR) is a preferred method for rapid and accurate quantification of gene expression studies. Appropriate application of RT-qPCR requires accurate normalization though the use of reference genes. As no single reference gene is universally suitable for all experiments, thus reference gene(s) validation under different experimental conditions is crucial for RT-qPCR analysis. To date, only a few studies on reference genes have been done in other plants but none in papaya. In the present work, we selected 21 candidate reference genes, and evaluated their expression stability in 246 papaya fruit samples using three algorithms, geNorm, NormFinder and RefFinder. The samples consisted of 13 sets collected under different experimental conditions, including various tissues, different storage temperatures, different cultivars, developmental stages, postharvest ripening, modified atmosphere packaging, 1-methylcyclopropene (1-MCP) treatment, hot water treatment, biotic stress and hormone treatment. Our results demonstrated that expression stability varied greatly between reference genes and that different suitable reference gene(s) or combination of reference genes for normalization should be validated according to the experimental conditions. In general, the internal reference genes EIF (Eukaryotic initiation factor 4A), TBP1 (TATA binding protein 1) and TBP2 (TATA binding protein 2) genes had a good performance under most experimental conditions, whereas the most widely present used reference genes, ACTIN (Actin 2), 18S rRNA (18S ribosomal RNA) and GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) were not suitable in many experimental conditions. In addition, two commonly used programs, geNorm and Normfinder, were proved sufficient for the validation. This work provides the first systematic analysis for the selection of superior reference genes for accurate transcript normalization in papaya under different experimental conditions.
To obtain reliable quantitative RT-PCR data, normalization relative to stable housekeeping genes is required. However, in practice, expression levels of 'typical' housekeeping genes have been found to vary between tissues and under different experimental conditions. To date, validation studies of reference genes in insects are extremely rare and have never been performed in locusts. In this study, putative housekeeping genes were identified in the desert locust, Schistocerca gregaria and two different software programs (geNorm and Normfinder) were applied to assess the stability of thesegenes.
We have identified seven orthologs of commonly used housekeeping genes in the desert locust. The selected genes were the orthologs of actin, EF1a, GAPDH, RP49, TubA1, Ubi, and CG13220. By employing real time RT-PCR we have analysed the expression of these housekeeping genes in brain tissue of fifth instar nymphs and adults. In the brain of fifth instar nymphs geNorm indicated Sg-EF1a, Sg-GAPDH and Sg-RP49 as most stable genes, while Normfinder ranked Sg-RP49, Sg-EF1a and Sg-ACT as most suitable candidates for normalization. The best normalization candidates for gene expression studies in the brains of adult locusts were Sg-EF1a, Sg-GAPDH and Sg-Ubi according to geNorm, while Normfinder determined Sg-GAPDH, Sg-Ubi and Sg-ACT as the most stable housekeeping genes.
To perform transcript profiling studies on brains of the desert locust, the use of Sg-RP49, Sg-EF1a and Sg-ACT as reference genes is proposed for studies of fifth instar nymphs. In experiments with adult brains, however, the most preferred reference genes were Sg-GAPDH, Sg-Ubi and Sg-EF1a. These data will facilitate transcript profiling studies in desert locusts and provide a good starting point for the initial selection of genes for validation studies in other insects.
Reference genes with stable expression are required to normalize expression differences of target genes in qPCR experiments. Several procedures and companion software have been proposed to find the most stable genes. Model based procedures are attractive because they provide a solid statistical framework. NormFinder, a widely used software, uses a model based method. The pairwise comparison procedure implemented in GeNorm is a simpler procedure but one of the most extensively used. In the present work a statistical approach based in Maximum Likelihood estimation under mixed models was tested and compared with NormFinder and geNorm softwares. Sixteen candidate genes were tested in whole blood samples from control and heat stressed sheep.
A model including gene and treatment as fixed effects, sample (animal), gene by treatment, gene by sample and treatment by sample interactions as random effects with heteroskedastic residual variance in gene by treatment levels was selected using goodness of fit and predictive ability criteria among a variety of models. Mean Square Error obtained under the selected model was used as indicator of gene expression stability. Genes top and bottom ranked by the three approaches were similar; however, notable differences for the best pair of genes selected for each method and the remaining genes of the rankings were shown. Differences among the expression values of normalized targets for each statistical approach were also found.
Optimal statistical properties of Maximum Likelihood estimation joined to mixed model flexibility allow for more accurate estimation of expression stability of genes under many different situations. Accurate selection of reference genes has a direct impact over the normalized expression values of a given target gene. This may be critical when the aim of the study is to compare expression rate differences among samples under different environmental conditions, tissues, cell types or genotypes. To select reference genes not only statistical but also functional and biological criteria should be considered. Under the method here proposed SDHA/MDH1 have arisen as the best set of reference genes to be used in qPCR assays to study heat shock in ovine blood samples.
The plastid transformation approach offers a number of unique advantages, including high-level transgene expression, multi-gene engineering, transgene containment, and a lack of gene silencing and position effects. The extension of plastid transformation technology to monocotyledonous cereal crops, including rice, bears great promise for the improvement of agronomic traits, and the efficient production of pharmaceutical or nutritional enhancement. Here, we report a promising step towards stable plastid transformation in rice. We produced fertile transplastomic rice plants and demonstrated transmission of the plastid-expressed green fluorescent protein (GFP) and aminoglycoside 3′-adenylyltransferase genes to the progeny of these plants. Transgenic chloroplasts were determined to have stably expressed the GFP, which was confirmed by both confocal microscopy and Western blot analyses. Although the produced rice plastid transformants were found to be heteroplastomic, and the transformation efficiency requires further improvement, this study has established a variety of parameters for the use of plastid transformation technology in cereal crops.
Cereal Crop; Chloroplast Genome; Mono-cotyledonous Plant Species; Plastid Transformation; Rice
Gene expression analysis by reverse transcriptase real-time or quantitative polymerase chain reaction (RT-qPCR) is becoming widely used for non-model plant species. Given the high sensitivity of this method, normalization using multiple housekeeping or reference genes is critical, and careful selection of these reference genes is one of the most important steps to obtain reliable results. In this study, reference genes commonly used for other plant species were investigated to identify genes displaying highly uniform expression patterns in different varieties, tissues, developmental stages, fungal infection, and osmotic stress conditions for the non-model crop Musa (banana and plantains). The expression stability of six candidate reference genes was tested on six different sample sets, and the results were analyzed using the publicly available algorithms geNorm and NormFinder. Our results show that variety, plant material, primer set, and gene identity can all influence the robustness and outcome of RT-qPCR analysis. In the case of Musa, a combination of three reference genes (EF1, TUB and ACT) can be used for normalization of gene expression data from greenhouse leaf samples. In the case of shoot meristem cultures, numerous combinations can be used because the investigated reference genes exhibited limited variability. In contrast, variability in expression of the reference genes was much larger among leaf samples from plants grown in vitro, for which the best combination of reference genes (L2 and ACT genes) is still suboptimal. Overall, our data confirm that the stability of candidate reference genes should be thoroughly investigated for each experimental condition under investigation.
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The online version of this article (doi:10.1007/s11032-012-9711-1) contains supplementary material, which is available to authorized users.
Musa; Banana; Reference genes; qPCR; Gene expression; Mycosphaerella
The Rice Proteome Database is the first detailed database to describe the proteome of rice. The current release contains 21 reference maps based on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of proteins from rice tissues and subcellular compartments. These reference maps comprise 11 941 identified proteins showing tissue and subcellular localization, corresponding to 4180 separate protein entries in the database. The Rice Proteome Database contains the calculated properties of each protein such as molecular weight, isoelectric point and expression; experimentally determined properties such as amino acid sequences obtained using protein sequencers and mass spectrometry; and the results of database searches such as sequence homologies. The database is searchable by keyword, accession number, protein name, isoelectric point, molecular weight and amino acid sequence, or by selection of a spot on one of the 2D-PAGE reference maps. Cross-references are provided to tools for proteomics and to other 2D-PAGE databases, which in turn provide many links to other molecular databases. The information in the Rice Proteome Database is updated weekly, and is available on the World Wide Web at http://gene64.dna.affrc.go.jp/RPD/.
• Background and Aims Glycinebetaine (GB), a quaternary ammonium compound, is a very effective compatible solute. In higher plants, GB is synthesized from choline (Cho) via betaine aldehyde (BA). The first and second steps in the biosynthesis of GB are catalysed by choline monooxygenase (CMO) and by betaine aldehyde dehydrogenase (BADH), respectively. Rice (Oryza sativa), which has two genes for BADH, does not accumulate GB because it lacks a functional gene for CMO. Rice plants accumulate GB in the presence of exogenously applied BA, which leads to the development of a significant tolerance to salt, cold and heat stress. The goal in this study was to evaluate and to discuss the effects of endogenously accumulated GB in rice.
• Methods Transgenic rice plants that overexpressed a gene for CMO from spinach (Spinacia oleracea) were produced by Agrobacterium-mediated transformation. After Southern and western blotting analysis, GB in rice leaves was quantified by 1H-NMR spectroscopy and the tolerance of GB-accumulating plants to abiotic stress was investigated.
• Key Results Transgenic plants that had a single copy of the transgene and expressed spinach CMO accumulated GB at the level of 0·29–0·43 μmol g−1 d. wt and had enhanced tolerance to salt stress and temperature stress in the seedling stage.
• Conclusions In the CMO-expressing rice plants, the localization of spinach CMO and of endogenous BADHs might be different and/or the catalytic activity of spinach CMO in rice plants might be lower than it is in spinach. These possibilities might explain the low levels of GB in the transgenic rice plants. It was concluded that CMO-expressing rice plants were not effective for accumulation of GB and improvement of productivity.
Oryza sativa; glycinebetaine; choline monooxygenase; transgenic rice; tolerance to abiotic stress
Value-added processing with respect to rice milling has traditionally treated the rice bran layer as a homogenous material that contains significant concentrations of high-value components of interest for pharmaceutical and nutraceutical applications. Investigators have shown that high-value components in the rice bran layer vary from differences in kernel-thickness, bran fraction, rice variety, and environmental conditions during the growing season. The objectives of this study were to quantify the amount of rice bran removed at pre-selected milling times and to correlate the amount of rice bran removed at each milling time with the concentration of vitamin E, gamma-oryzanol, rice bran saccharide, and protein obtained. The ultimate goal of this research is to show that rice bran fractionation is a useful method to obtain targeted, nutrient-rich bran samples for value-added processing. Two long grain rice cultivars, Cheniere and Cypress, were milled at discrete times between 3 and 40 seconds using a McGill mill to obtain bran samples for analysis. Results showed that the highest oryzanol and protein concentrations were found in the outer portion of the rice bran layer, while the highest rice bran saccharide concentration was found in the inner portion of the bran layer. Vitamin E concentration showed no significant difference across the bran layer within a variety, though the highest magnitude of concentration occurs within the first 10 seconds of milling for both varieties. To extract the higher concentration of oryzanol and protein only the outer portion of the bran layer requires processing, while to extract the higher concentration of rice bran saccharide, only the inner portion of the bran layer requires processing. Rice bran fractionation allows for the selective use of portions of the bran layer and is advantageous for two reasons: (1) bran fractions contain higher concentrations of components of interest with respect to the overall bran layer average, and (2) less bran needs to be processed to obtain components of interest.
Black rice is rich in anthocyanin and is expected to have more healthful dietary potential than white rice. We assessed expression of anthocyanin in black rice cultivars using a newly designed 135 K Oryza sativa microarray. A total of 12,673 genes exhibited greater than 2.0-fold up- or down-regulation in comparisons between three rice cultivars and three seed developmental stages. The 137 transcription factor genes found to be associated with production of anthocyanin pigment were classified into 10 groups. In addition, 17 unknown and hypothetical genes were identified from comparisons between the rice cultivars. Finally, 15 out of the 17 candidate genes were verified by RT-PCR analysis. Among the genes, nine were up-regulated and six exhibited down-regulation. These genes likely play either a regulatory role in anthocyanin biosynthesis or are related to anthocyanin metabolism during flavonoid biosynthesis. While these genes require further validation, the results here underline the potential use of the new microarray and provide valuable insight into anthocyanin pigment production in rice.
Anthocyanin; Black rice; Pigmentation; Rice microarray; Transcription factor
ClpB-cyt/HSP100 protein acts as chaperone, mediating disaggregation of denatured proteins. Previous studies have shown that ClpB-cyt/HSP100 gene belongs to the group class I Clp ATPase proteins and ClpB-cyt/HSP100 transcript is regulated by heat stress and developmental cues.
Nine ORFs were noted to constitute rice class I Clp ATPases in the following manner: 3 ClpB proteins (ClpB-cyt, Os05g44340; ClpB-m, Os02g08490; ClpB-c, Os03g31300), 4 ClpC proteins (ClpC1, Os04g32560; ClpC2, Os12g12580; ClpC3, Os11g16590; ClpC4, Os11g16770) and 2 ClpD proteins (ClpD1, Os02g32520; ClpD2, Os04g33210). Using the respective signal sequences cloned upstream to GFP/CFP reporter proteins and transient expression studies with onion epidermal cells, evidence is provided that rice ClpB-m and Clp-c proteins are indeed localized to their respective cell locations mitochondria and chloroplasts, respectively. Associated with their diverse cell locations, domain structures of OsClpB-c, OsClpB-m and OsClpB-cyt proteins are noted to possess a high-level conservation. OsClpB-cyt transcript is shown to be enriched at milk and dough stages of seed development. While expression of OsClpB-m was significantly less as compared to its cytoplasmic and chloroplastic counterparts in different tissues, this transcript showed highest heat-induced expression amongst the 3 ClpB proteins. OsClpC1 and OsClpC2 are predicted to be chloroplast-localized as is the case with all known plant ClpC proteins. However, the fact that OsClpC3 protein appears mitochondrial/chloroplastic with equal probability and OsClpC4 a plasma membrane protein reflects functional diversity of this class. Different class I Clp ATPase transcripts were noted to be cross-induced by a host of different abiotic stress conditions. Complementation assays of Δhsp104 mutant yeast cells showed that OsClpB-cyt, OsClpB-m, OsClpC1 and OsClpD1 have significantly positive effects. Remarkably, OsClpD1 gene imparted appreciably high level tolerance to the mutant yeast cells.
Rice class I Clp ATPase gene family is constituted of 9 members. Of these 9, only 3 belonging to ClpB group are heat stress regulated. Distribution of ClpB proteins to different cell organelles indicates that their functioning might be critical in different cell locations. From the complementation assays, OsClpD1 appears to be more effective than OsClpB-cyt protein in rescuing the thermosensitive defect of the yeast ScΔhsp104 mutant cells.
A new glutelin gene, designated GluD-1, has been discovered by comparing the seed storage proteins from 48 japonica and indica rice cultivars on SDS-PAGE gels. Evidence that GluD-1 is a member of the glutelin family was provided by Western blots using anti-glutelin antiserum and by mapping the gene to the chromosomal glutelin gene cluster. The limited GluD-1 size polymorphism among the rice varieties is due to amino acid substitutions rather than to post-transcriptional modification. GluD-1 is maximally expressed in the starchy endosperm starting at 5 d after flowering (DAF) and increasing through 30 DAF, a major difference from the other glutelins which are primarily expressed in the subaleurone from 10–16 DAF. Only about 0.2 kb of the GluD-1 promoter was sufficient to confer inner starchy endosperm-specific expression. The 0.2 kb truncated GluD-1 promoter contains a bifactorial endosperm box consisting of a truncated GCN4 motif (TGA(G/C)TCA) and AAAG Prolamin box (P box), and ACGT and AACA motifs as cis-regulatory elements. Gel retardation assays and trans-activation experiments indicated that the truncated GCN4 and P box are specifically recognized by RISBZ1 b-ZIP and RPBF Dof activators in vitro, respectively, and are synergistically transactivated, indicating that combinatorial interactions of these motifs are involved in essential endosperm-specific regulation. Furthermore, deviation from the cognate GCN4 motif alters tissue-specific expression in the inner starchy endosperm to include other endosperm tissues.
bZIP; DOF; endosperm specific expression; glutelin; promoter; rice; seed storage protein; transgenic crop
Few studies have focused on the expression of heat shock proteins (HSPs) after chronic heat stress. The objective of this study was to investigate the effect of chronic high temperature–humidity index treatment on the expressions of HSP60, HSP70, HSP90, HSPA2 and HSC70, in the Rex rabbit testis and the expressions of these proteins after recovery from the chronic heat shock. Thirty mature male rabbits of the same age were randomly divided into three groups: control, heat stress, and recovery. The western blot results showed that the expressional levels of HSP60, HSP90, and HSC70 increased significantly and HSPA2 was elevated slightly after a 9-week heat treatment. HSP70 was absent in the control testis and had a high level of expression after heat stress. All of these proteins partially reverted back to normal levels after a 9-week recovery. The immunohistochemical results indicated that the expression patterns of HSP60, HSP90, HSPA2, and HSC70 did not change.
Heat stress; Male Rex rabbit; Testis; HSPs
The dog is frequently used as a model for hematologic human diseases. In this study the suitability of nine potential reference genes for quantitative RT-PCR studies in canine whole blood was investigated.
The expression of these genes was measured in whole blood samples of 263 individual dogs, representing 73 different breeds and a group of 40 mixed breed dogs, categorized into healthy dogs and dogs with internal and hematological diseases, and dogs that underwent a surgical procedure. GeNorm analysis revealed that a combination of 5 to 6 of the most stably expressed genes constituted a stable normalizing factor. Evaluation of the expression revealed different ranking of reference genes in Normfinder and GeNorm. The disease category and the white blood cell count significantly affected reference gene expression.
The discrepancy between the ranking of reference genes in this study by Normfinder and Genorm can be explained by differences between the experimental groups such as "disease category" and "WBC count". This stresses the importance of assessing the expression stability of potential reference genes for gene experiments in canine whole blood anew for each specific experimental condition.
Gene expression studies are a prerequisite for understanding the biological function of genes. Because of its high sensitivity and easy use, quantitative PCR (qPCR) has become the gold standard for gene expression quantification. To normalise qPCR measurements between samples, the most prominent technique is the use of stably expressed endogenous control genes, the so called reference genes. However, recent studies show there is no universal reference gene for all biological questions. Roses are important ornamental plants for which there has been no evaluation of useful reference genes for gene expression studies.
We used three different algorithms (BestKeeper, geNorm and NormFinder) to validate the expression stability of nine candidate reference genes in different rose tissues from three different genotypes of Rosa hybrida and in leaves treated with various stress factors. The candidate genes comprised the classical "housekeeping genes" (Actin, EF-1α, GAPDH, Tubulin and Ubiquitin), and genes showing stable expression in studies in Arabidopsis (PP2A, SAND, TIP and UBC). The programs identified no single gene that showed stable expression under all of the conditions tested, and the individual rankings of the genes differed between the algorithms. Nevertheless the new candidate genes, specifically, PP2A and UBC, were ranked higher as compared to the other traditional reference genes. In general, Tubulin showed the most variable expression and should be avoided as a reference gene.
Reference genes evaluated as suitable in experiments with Arabidopsis thaliana were stably expressed in roses under various experimental conditions. In most cases, these genes outperformed conventional reference genes, such as EF1-α and Tubulin. We identified PP2A, SAND and UBC as suitable reference genes, which in different combinations may be used for normalisation in expression analyses via qPCR for different rose tissues and stress treatments. However, the vast genetic variation found within the genus Rosa, including differences in ploidy levels, might also influence expression stability of reference genes, so that future research should also consider different genotypes and ploidy levels.
Colorectal cancer (CRC) is a major public health issue worldwide, and novel tumor markers may contribute to its efficient management by helping in early detection, prognosis or surveillance of disease. The aim of our study was to identify new serum biomarkers for CRC, and we followed a phased biomarker discovery and validation process to obtain an accurate preliminary assessment of potential clinical utility. We compared colonic tumors and matched normal tissue from 15 CRC patients, using two-dimensional difference gel electrophoresis (2D-DIGE), and identified 17 proteins that had significant differential expression. These results were further confirmed by western blotting for heat shock protein (HSP) 60, glutathione-S-transferase Pi, α-enolase, T-complex protein 1 subunit β, and leukocyte elastase inhibitor, and by immunohistochemistry for HSP60. Using mAbs raised against HSP60, we developed a reliable (precision of 5–15%) and sensitive (0.3 ng·mL−1) immunoassay for the detection of HSP60 in serum. Elevated levels of HSP60 were found in serum from CRC patients in two independent cohorts; the receiver-operating characteristic curve obtained in 112 patients with CRC and 90 healthy controls had an area under the curve (AUC) of 0.70, which was identical to the AUC of carcinoembryonic antigen. Combination of serum markers improved clinical performance: the AUC of a three-marker logistic regression model combining HSP60, carcinoembryonic antigen and carbohydrate antigen 19-9 reached 0.77. Serum HSP60 appeared to be more specific for late-stage CRC; therefore, future studies should evaluate its utility for determining prognosis or monitoring therapy rather than early detection.
2D-DIGE; colorectal cancer; HSP60; marker validation; serum biomarker
Heat shock proteins (Hsps) constitute an important component in the heat shock response of all living systems. Among the various plant Hsps (i.e. Hsp100, Hsp90, Hsp70 and Hsp20), Hsp20 or small Hsps (sHsps) are expressed in maximal amounts under high temperature stress. The characteristic feature of the sHsps is the presence of α-crystallin domain (ACD) at the C-terminus. sHsps cooperate with Hsp100/Hsp70 and co-chaperones in ATP-dependent manner in preventing aggregation of cellular proteins and in their subsequent refolding. Database search was performed to investigate the sHsp gene family across rice genome sequence followed by comprehensive expression analysis of these genes.
We identified 40 α-crystallin domain containing genes in rice. Phylogenetic analysis showed that 23 out of these 40 genes constitute sHsps. The additional 17 genes containing ACD clustered with Acd proteins of Arabidopsis. Detailed scrutiny of 23 sHsp sequences enabled us to categorize these proteins in a revised scheme of classification constituting of 16 cytoplasmic/nuclear, 2 ER, 3 mitochondrial, 1 plastid and 1 peroxisomal genes. In the new classification proposed herein nucleo-cytoplasmic class of sHsps with 9 subfamilies is more complex in rice than in Arabidopsis. Strikingly, 17 of 23 rice sHsp genes were noted to be intronless. Expression analysis based on microarray and RT-PCR showed that 19 sHsp genes were upregulated by high temperature stress. Besides heat stress, expression of sHsp genes was up or downregulated by other abiotic and biotic stresses. In addition to stress regulation, various sHsp genes were differentially upregulated at different developmental stages of the rice plant. Majority of sHsp genes were expressed in seed.
We identified twenty three sHsp genes and seventeen Acd genes in rice. Three nucleocytoplasmic sHsp genes were found only in monocots. Analysis of expression profiling of sHsp genes revealed that these genes are differentially expressed under stress and at different stages in the life cycle of rice plant.
Rhizoctonia solani is a major fungal pathogen of rice (Oryza sativa L.) that causes great yield losses in all rice-growing regions of the world. Here we report the draft genome sequence of the rice sheath blight disease pathogen, R. solani AG1 IA, assembled using next-generation Illumina Genome Analyser sequencing technologies. The genome encodes a large and diverse set of secreted proteins, enzymes of primary and secondary metabolism, carbohydrate-active enzymes, and transporters, which probably reflect an exclusive necrotrophic lifestyle. We find few repetitive elements, a closer relationship to Agaricomycotina among Basidiomycetes, and expand protein domains and families. Among the 25 candidate pathogen effectors identified according to their functionality and evolution, we validate 3 that trigger crop defence responses; hence we reveal the exclusive expression patterns of the pathogenic determinants during host infection.
The rice sheath blight pathogen, Rhizoctonia solani, is an important fungal pathogen that can devastate rice and maize crops. Zheng and colleagues sequence and assemble the R. solani AG1 IA genome—the first to be sequenced from the Rhizoctonia genus—using Illumina sequencing technology.