Real-time Expression Analysis Research Articles

Gene Regulatory Networks in Peripheral Mononuclear Cells Reveals Critical Regulatory Modules and Regulators of Multiple Sclerosis

Multiple sclerosis (MS) is a complex, demyelinating disease with the involvement of autoimmunity and neurodegeneration. Increasing efforts have been made towards identifying the diagnostic markers to differentiate the classes of MS from other similar neurological conditions. Using a systems biology approach, we constructed four types of gene regulatory networks (GRNs) involved in peripheral blood mononuclear cells (PBMCs). The regulatory strength of each GRN across primary progressive MS (PPMS), relapsing-remitting MS (RRMS), secondary progressive MS (SPMS), and control were evaluated by an integrity algorithm. Among the constructed GRNs (referred as TF_gene_miRNA), POU3F2_CDK6_hsa-miR-590-3p, MEIS1_CASC3_hsa-miR-1261, STAT3_OGG1_hsa-miR-298, and TCF4_FMR1_hsa-miR-301b were top-ranked and differentially regulated in all classes of MS compared to control. These GRNs showed potential involvement in regulating various molecular pathways such as interleukin, integrin, glypican, sphingosine phosphate, androgen, and Wnt signaling pathways. For validation, the qPCR analysis of the GRN components (TFs, gene, and miRNAs) in PBMCs of healthy controls (n = 30), RRMS (n = 14), PPMS (n = 13) and SPMS (n = 12) were carried out. Real-time expression analysis of GRNs showed a similar regulatory pattern as derived from our systems biology approach. Also, our study provided several novel GRNs that regulate unique and common molecular mechanisms between MS conditions. Hence, these regulatory components of GRNs will help to understand the disease mechanism across MS classes and further insight may though light towards diagnosis.

A comprehensive analysis of the B3 superfamily identifies tissue-specific and stress-responsive genes in chickpea (Cicer arietinum L.).

The aim of this study was to provide a comprehensive analysis of the plant-specific B3 domain-containing transcription factors (TFs) in chickpea. Scanning of the chickpea genome resulted in the identification of 51 B3 domain-containing TFs that were located on seven out of eight chickpea chromosomes. Based on the presence of additional domains other than the B3 domain, the candidates were classified into four subfamilies, i.e., ARF (24), REM (19), LAV (6) and RAV (2). Phylogenetic analysis classified them into four groups in which members of the same group had similar intron-exon organization and motif composition. Genome duplication analysis of the candidate B3 genes revealed an event of segmental duplication that was instrumental in the expansion of the B3 gene family. Ka/Ks analysis showed that the B3 gene family was under purifying selection. Further, chickpea B3 genes showed maximum orthology with Medicago followed by soybean and Arabidopsis. Promoter analyses of the B3 genes led to the identification of several tissue-specific and stress-responsive cis-regulatory elements. Expression profiling of the candidate B3 genes using publicly available RNA-seq data of several chickpea tissues indicated their putative role in plant development and abiotic stress response. These findings were further validated by real-time expression analysis. Overall, this study provides a comprehensive analysis of the B3 domain-containing proteins in chickpea that would aid in devising strategies for crop manipulation in chickpea.

Genome-Wide Identification, Expression Analysis, and Subcellular Localization of Carthamus tinctorius bHLH Transcription Factors.

The basic helix–loop–helix (bHLH) family is the second largest superfamily of transcription factors that belongs to all three eukaryotic kingdoms. The key function of this superfamily is the regulation of growth and developmental mechanisms in plants. However, the bHLH gene family in Carthamus tinctorius has not yet been studied. Here, we identified 41 bHLH genes in Carthamus tinctorius that were classified into 23 subgroups. Further, we conducted a phylogenetic analysis and identified 10 conserved protein motifs found in the safflower bHLH family. We comprehensively analyzed a group of bHLH genes that could be associated with flavonoid biosynthesis in safflower by gene expression analysis, gene ontology annotation, protein interaction network prediction, subcellular localization of the candidate CtbHLH40 gene, and real-time quantitative expression analysis. This study provides genome-wide identification of the genes related to biochemical and physiological processes in safflower.

Soil salinity and mechanical obstruction differentially affects embryonic root architecture in different rice genotypes from West Bengal

Making the first contact with the soil environment, roots are the first responders of soil borne stress including nutrient and water scarcity, hyper salinity and soil compactness. Embryonic root responds by bending, twisting, foraging and regulation of lateral root initiation. Traditional rice cultivars adapted to diverse topography harbour novel stress tolerance traits. We established a quantitative measurement coefficient termed stress adaptation coefficient (SAC) dependent on root responses to stress and applied it to study the effect of mechanical stress and salinity on roots of rice genotypes collected all across West Bengal, India. The responses of root to mechanical and salinity stress are overlapping. Analysis of the response of roots by means of SAC in 28 rice cultivars including high-yield salt tolerant varieties as well as geographically isolated native rice genotypes shows that many of the salt tolerant varieties also perform better in mechanical stress while the opposite is not always true. Transcriptome analysis through cDNA microarray of stress sensitive variety IR64 shows about 6000 common transcripts to be differentially regulated among the two stresses. Quantitative real time expression analyses of salt sensitive and known salt tolerant varieties reveal the involvement of identified genes in salinity and mechanical stress. Overall, our study indicates that there is an important commonality in the molecular basis of salt and mechanical stress and presents an easy-to-perform early establishment stress screen for rice genotypes.

HvWRKY23 regulates flavonoid glycoside and hydroxycinnamic acid amide biosynthetic genes in barley to combat Fusarium head blight.

Crop plant resistance against pathogens is governed by dynamic molecular and biochemical responses driven by complex transcriptional networks. However, the underlying mechanisms are largely unclear. Here we report an interesting role of HvWRKY23 transcription factor (TF) in modulating defense response against Fusarium head blight (FHB) in barley. The combined approach of gene silencing, metabolomics, real time expression analysis and ab initio bioinformatics tools led to the identification of the HvWRKY23 role in FHB resistance. The knock-down of HvWRKY23 gene in the FHB resistant barley genotype CI9831, followed by inoculation with Fusarium graminearum, led to the down regulation of key flavonoid and hydroxycinnamic acid amide biosynthetic genes resulting in reduced accumulation of resistant related (RR) secondary metabolites such as pelargonidin 3-rutinoside, peonidin 3-rhamnoside-5-glucoside, kaempferol 3-O-arabinoside and other flavonoid glycosides. Reduced abundances of RR metabolites in TF silenced plants were also associated with an increased proportion of spikelets diseased and amount of fungal biomass in spikelets, depicting the role of HvWRKY23 in disease resistance. The luciferase reporter assay demonstrated binding of HvWRKY23 protein to promoters of key flavonoid and hydroxycinnamic acid amides (HCAA)biosynthetic genes, such as HvPAL2, HvCHS1, HvHCT, HvLAC15 and HvUDPGT. The accumulation of high abundances of HCAAs and flavonoid glycosides reinforce cell walls to contain the pathogen to initial infection area. This gene in commercial cultivars can be edited, if non-functional, to enhance resistance against FHB.

English

The expression profile of pathogenesis related genes are signatures of an infection response in plant cells. Pathogenic infections can increase or reduce gene expression in a plant system in a relatively specific pattern. These expression patterns can be used as standards in pathogenicity studies and, where phenotypic expression is normally used to gauge a plants response to infection, it could additionally present a more rapid and early screening reference tool. Three genes: catalase (CAT), pathogen related protein (PR10), and phenylalanine ammonia (PAL) all implicitly implicated in the plant disease response pathway were targeted for analysis during the infection of Fusarium oxysporum f. sp. cubense tropical race 4 (FOCR4) in banana Musa acuminata cv. Berangan seedlings after a standard challenge under growth room conditions. Distinct patterns of gene expression were observed at three infection time points by real time expression analysis. There was a sequential 10-fold reduction in expression for the PR gene while, the PAL and CAT genes were both upregulated. These results present a set of reference genes that could be used for screening of a plant’s response to Fusarium before the onset of symptoms

Cervical cancer subtypes harbouring integrated and/or episomal HPV16 portray distinct molecular phenotypes based on transcriptome profiling of mRNAs and miRNAs

Heterogeneity in cervical cancers (CaCx) in terms of HPV16 physical status prompted us to investigate the mRNA and miRNA signatures among the different categories of CaCx samples. We performed microarray-based mRNA expression profiling and quantitative real-time PCR-based expression analysis of some prioritised miRNAs implicated in cancer-related pathways among various categories of cervical samples. Such samples included HPV16-positive CaCx cases that harboured either purely integrated HPV16 genomes (integrated) and those that harboured episomal viral genomes, either pure or concomitant with integrated viral genomes (episomal), which were compared with normal cervical samples that were either HPV negative or positive for HPV16. The mRNA expression profile differed characteristically between integrated and episomal CaCx cases for enriched biological pathways. miRNA expression profiles also differed among CaCx cases compared with controls (upregulation—miR-21, miR-16, miR-205, miR-323; downregulation—miR-143, miR-196b, miR-203, miR-34a; progressive upregulation—miR-21 and progressive downregulation—miR-143, miR-34a, miR-196b and miR-203) in the order of HPV-negative controls, HPV16-positive non-malignant samples and HPV16-positive CaCx cases. miR-200a was upregulated in HPV16-positive cervical tissues irrespective of histopathological status. Expression of majority of the predicted target genes was negatively correlated with their corresponding miRNAs, irrespective of the CaCx subtypes. E7 mRNA expression correlated positively with miR-323 expression among episomal cases and miR-203, among integrated cases. miR-181c expression was downregulated only among the episomal CaCx cases and negatively correlated with protein coding transcript of the proliferative target gene, CKS1B of the significantly enriched “G2/M DNA Damage Checkpoint Regulation” pathway among CaCx cases. Thus, the two CaCx subtypes are distinct entities at the molecular level, which could be differentially targeted for therapy. In fact, availability of a small molecule inhibitor of CKS1B, suggests that drugging CKS1B could be a potential avenue of treating the large majority of CaCx cases harbouring episomal HPV16.

Promoter of the wheat lipid transfer protein, TdLTP4, drives leaf-preferential expression in transgenic Arabidopsis plants.

In a previous report, a gene encoding a durum wheat lipid transfer protein, TdLTP4, was characterised as induced by abiotic and biotic stresses. In the present work, we investigated the regulation of the gene TdLTP4. A TdLTP4 promoter (PrTdLTP4) region of around 868-bp was isolated and sequenced. Its analysis revealed the presence of several DNA boxes known to be important mainly in the regulation of genes expressed under abiotic stress (salt and dehydration), abscisic acid (ABA) and pathogen responsiveness. The whole PrTdLTP4 fragment was fused to the reporter gene β-glucuronidase (gusA) and analysed in transgenic Arabidopsis plants. Histochemical assays of transgenic Arabidopsis plants showed that the 868-bp fragment of TdLTP4 gene promoter was found to be sufficient for both spatial and temporal patterns of its expression. Under control conditions, GUS histochemical staining was observed significantly only in young leaves of 8- and 12-day-old plants. Whereas after stress challenge especially with NaCl and mannitol, GUS transcripts expression increased substantially in leaves of 30-day-old transgenic seedlings. Real-time qPCR expression analysis of the gusA gene, confirmed the results of histochemical assays. Taken together these data provide evidence that PrTdLTP4 functions as abiotic-stress-inducible promoter in a heterologous dicot system and could be an excellent tool for future crop improvement.

Non-steroidal anti-inflammatory drugs, acetaminophen and ibuprofen, induce phenotypic antibiotic resistance in Escherichia coli: roles of marA and acrB.

The factors contributing to antibiotic resistance in bacteria are an important area of study. Sodium salicylate (NaSal), a non-steroidal anti-inflammatory drug (NSAID), increases antibiotic resistance by inducing the expression of MarA, a transcription factor, which increases the AcrAB-TolC efflux pump. MarA is a substrate of Lon protease and the Δlon strain displays a high degree of antibiotic resistance. This study was initiated to identify commonly used NSAIDs that may induce antibiotic resistance and to compare their efficacies with NaSal and acetyl salicylic acid (ASA). Quantitative real-time expression analysis revealed induction of marA and acrB by NaSal, ASA, acetaminophen (APAP) and ibuprofen. Further, dose studies demonstrated that NaSal and ASA induce resistance at ∼2 mM while APAP and ibuprofen induce resistance at ∼5-10 mM. To dissect the roles of key molecules, atomic force microscopy and functional studies were performed using WT, Δlon, ΔmarA, ΔacrB, ΔlonΔmarA and ΔlonΔacrB strains. The induction of antibiotic resistance by NaSal, ASA and APAP is relatively higher and is partly dependent on marA, whereas ibuprofen which induces lower antibiotic resistance shows complete marA dependence. Notably, NaSal, ASA, APAP and ibuprofen induce antibiotic resistance in an acrB-dependent manner. The possible significance of some NSAIDs in inducing antibiotic resistance is discussed.

Genome-wide transcriptome analysis using RNA-Seq reveals a large number of differentially expressed genes in a transient MCAO rat model

BackgroundThe transient middle cerebral artery occlusion (tMCAO) model is used for studying the molecular mechanisms of ischemic damage and neuroprotection. Numerous studies have demonstrated the role of individual genes and associated signaling pathways in the pathogenesis of ischemic stroke. Here, the tMCAO model was used to investigate the genome-wide response of the transcriptome of rat brain tissues to the damaging effect of ischemia and subsequent reperfusion.ResultsMagnetic resonance imaging and histological examination showed that the model of focal ischemia based on endovascular occlusion of the right middle cerebral artery for 90 min using a monofilament, followed by restoration of the blood flow, led to reproducible localization of ischemic damage in the subcortical structures of the brain. High-throughput RNA sequencing (RNA-Seq) revealed the presence of differentially expressed genes (DEGs) in subcortical structures of rat brains resulting from hemisphere damage by ischemia after tMCAO, as well as in the corresponding parts of the brains of sham-operated animals. Real-time reverse transcription polymerase chain reaction expression analysis of 20 genes confirmed the RNA-Seq results. We identified 469 and 1939 genes that exhibited changes in expression of > 1.5-fold at 4.5 and 24 h after tMCAO, respectively. Interestingly, we found 2741 and 752 DEGs under ischemia–reperfusion and sham-operation conditions at 24 h vs. 4.5 h after tMCAO, respectively. The activation of a large number of genes involved in inflammatory, immune and stress responses, apoptosis, ribosome function, DNA replication and other processes was observed in ischemia–reperfusion conditions. Simultaneously, massive down-regulation of the mRNA levels of genes involved in the functioning of neurotransmitter systems was recorded. A Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that dozens of signaling pathways were associated with DEGs in ischemia–reperfusion conditions.ConclusionsThe data obtained revealed a global profile of gene expression in the rat brain sub-cortex under tMCAO conditions that can be used to identify potential therapeutic targets in the development of new strategies for the prevention and treatment of ischemic stroke.

Characterization of quorum sensing system in Clostridium chauvoei

Clostridium chauvoei causes fatal black quarter infection in cattle and buffaloes. The quorum sensing (QS) system, a bacterial cell to cell communication process, of the pathogen was characterized in the current study. The results indicated that C. chauvoei lacked luxS (autoinducer-2) based quorum sensing as detected by the sensor strain Vibrio harveyi BB170. This was supported by absence of luxS gene in C. chauvoei genome. However, the genomic analysis indicated the presence of agrBD system in all three genomes of C. chauvoei available at the NCBI database. The AgrD, which synthesizes QS messenger auto-inducing peptide, was a 44 amino acid protein which shared 59% identity and 75% similarity with AgrD of C. perfringens strain 13 and 56% identity (20% coverage) with Staphylococcus aureus N315. The functional cysteine amino acid was conserved in all the strains. The genomic organisation further suggests the presence of diguanylate cyclase, a gene responsible for synthesis of secondary messenger cyclic di-GMP, at 3’ immediate downstream of agrD gene. The real time expression analysis for agrD gene indicated that expression was better at 37 °C (1.9–3.7 fold increase) compared to a higher temperature of 40 °C. However, stable expression was observed at different growth stages (log and early stationary phase) with 0.8–1.4 fold changes in expression pattern. The results indicate the presence of a constitutively expressed agrBD quorum sensing system in C. chauvoei.

Synergy of clavine alkaloid ‘chanoclavine’ with tetracycline against multi-drug-resistant E. coli

The emergence of multi drug resistance (MDR) in Gram-negative bacteria (GNB) and lack of novel classes of antibacterial agents have raised an immediate need to identify antibacterial agents, which can reverse the phenomenon of MDR. The purpose of present study was to evaluate synergy potential and understanding the drug resistance reversal mechanism of chanoclavine isolated from Ipomoea muricata against the multi-drug-resistant clinical isolate of Escherichia coli (MDREC). Although chanoclavine did not show antibacterial activity of its own, but in combination, it could reduce the minimum inhibitory concentration (MIC) of tetracycline (TET) up to 16-folds. Chanoclavine was found to inhibit the efflux pumps which seem to be ATPase-dependent. In real-time expression analysis, chanoclavine showed down-regulation of different efflux pump genes and decreased the mutation prevention concentration of tetracycline. Further, in silico docking studies revealed significant binding affinity of chanoclavine with different proteins known to be involved in drug resistance. In in silico ADME/toxicity studies, chanoclavine was found safe with good intestinal absorption, aqueous solubility, medium blood–brain barrier (BBB), no CYP 2D6 inhibition, no hepatotoxicity, no skin irritancy, and non-mutagenic indicating towards drug likeliness of this molecule. Based on these observations, it is hypothesized that chanoclavine might be inhibiting the efflux of tetracycline from MDREC and thus enabling the more availability of tetracycline inside the cell for its action.

A Comparative Study of Host Response against Macrophomina phaseolina Infection in Sensitive and Resistant Jute (Corchorus sp) Species Unravels a Possible Defense Mechanism

Plants are inherently able to save themselves from any unfavorable conditions. However, they fail to exert ample response when encountered with pathogens like Macrophomina phaseolina which infects more than 500 crops. Wild plant species empowered by superlative mechanisms can conquest deadly pathogens, an example of which is C. trilocularis, a jute plant, resistant to this necrotrophic fungal pathogen. This study tried to ascertain the underlying resistance mechanism regarding the systemic acquired resistance (SAR) and cell wall fortification. Identification and characterization of genes involved in the systemic acquired resistant pathway implicated an activity of these genes with a basal expression in resistant species but irregular in the sensitive one (C. olitorius). Real-time expression analysis of genes involved in the synthesis of cell wall components like cellulose, hemicellulose, and lignin along with chemical and microscopic examinations signified preeminent lignification in C. trilocularis. However, this vital plant cell wall component was found to decrease in C. olitorius. The present investigation provided a deep insight into the defense strategy employed by the two species of jute at the molecular level. This study is expected to help in improving the fungal resistance of susceptible but farmer popular jute species. The same should be applicable to other vulnerable crops as well. Understanding how the pathogen is recognized, and an effective defensive response mounted, could ultimately lead to the development of novel strategies for providing durable plant resistance.

Activation-tagging in indica rice identifies a novel transcription factor subunit, NF-YC13 associated with salt tolerance

Nuclear factor Y (NF-Y) is a heterotrimeric transcription factor with three distinct NF-YA, NF-YB and NF-YC subunits. It plays important roles in plant growth, development and stress responses. We have reported earlier on development of gain-of-function mutants in an indica rice cultivar, BPT-5204. Now, we screened 927 seeds from 70 Ac/Ds plants for salinity tolerance and identified one activation-tagged salt tolerant DS plant (DS-16, T3 generation) that showed enhanced expression of a novel ‘histone-like transcription factor’ belonging to rice NF-Y subfamily C and was named as OsNF-YC13. Localization studies using GFP-fusion showed that the protein is localized to nucleus and cytoplasm. Real time expression analysis confirmed upregulation of transcript levels of OsNF-YC13 during salt treatment in a tissue specific manner. Biochemical and physiological characterization of the DS-16 revealed enhanced K+/Na+ ratio, proline content, chlorophyll content, enzymes with antioxidant activity etc. DS-16 also showed transcriptional up-regulation of genes that are involved in salinity tolerance. In-silico analysis of OsNF-YC13 promoter region evidenced the presence of various key stress-responsive cis-regulatory elements. OsNF-YC13 subunit alone does not appear to have the capacity for direct transcription activation, but appears to interact with the B- subunits in the process of transactivation.

Modulation of proline metabolic gene expression in Arabidopsis thaliana under water-stressed conditions by a drought-mitigating Pseudomonas putida strain

Although amelioration of drought stress in plants by plant growth promoting rhizobacteria (PGPR) is a well reported phenomenon, the molecular mechanisms governing it are not well understood. We have investigated the role of a drought ameliorating PGPR strain, Pseudomonas putida GAP-P45 on the regulation of proline metabolic gene expression in Arabidopsis thaliana under water-stressed conditions. Indeed, we found that Pseudomonas putida GAP-P45 alleviates the effects of water-stress in A. thaliana by drastic changes in proline metabolic gene expression profile at different time points post stress induction. Quantitative real-time expression analysis of proline metabolic genes in inoculated plants under water-stressed conditions showed a delayed but prolonged up-regulation of the expression of genes involved in proline biosynthesis, i.e., ornithine-Δ-aminotransferase (OAT), Δ 1 -pyrroline-5-carboxylate synthetase1 (P5CS1), Δ 1 -pyrroline-5-carboxylate reductase (P5CR), as well as proline catabolism, i.e., proline dehydrogenase1 (PDH1) and Δ 1 -pyrroline-5-carboxylate dehydrogenase (P5CDH). These observations were positively correlated with morpho-physiological evidences of water-stress mitigation in the plants inoculated with Pseudomonas putida GAP-P45 that showed better growth, increased fresh weight, enhanced plant water content, reduction in primary root length, enhanced chlorophyll content in leaves, and increased accumulation of endogenous proline. Our observations point towards PGPR-mediated enhanced proline turnover rate in A. thaliana under dehydration conditions.

Real-Time Expression Analysis of Selected Anticarsia gemmatalis multiple nucleopolyhedrovirus Gene Promoters during Infection of Permissive, Semipermissive and Nonpermissive Cell Lines.

Baculovirus infection follows a transcriptionally controlled sequence of gene expression that occurs by activation of different viral gene promoter sequences during infection. This sequence of promoter activation may be disrupted by cellular defenses against viral infection, which might interfere with viral progeny formation. In this work, the activity of the ie1, gp64, lef-1, vp39, p6.9 and polh promoters of the Anticarsia gemmatalis multiple nucleopolyhedrovirus was assessed during infection of permissive, semipermissive and nonpermissive cell lines by a novel methodology that detects reporter protein luminescence in real-time. This technique allowed us to characterize in rich detail the AgMNPV promoters in permissive cell lines and revealed differential profiles of expression in cells with limited permissivity that correlate well with limitations in viral DNA replication. Semipermissive and nonpermissive cell lines presented delays and restrictions in late and very late promoter expression. Cells undergoing apoptosis did not inhibit late gene expression; however, viral progeny formation is severely affected. This work demonstrates the application of the real-time luminescence detection methodology and how the promoter expression profile may be used to diagnose cellular permissivity to baculovirus infection.

Molecular phylogenomic study and the role of exogenous spermidine in the metabolic adjustment of endogenous polyamine in two rice cultivars under salt stress

Compelling evidences anticipated the well acclamation of involvement of exogenous and endogenous polyamines (PAs) in conferring salt tolerance in plants. Intracellular PA's anabolism and catabolism should have contributed to maintain endogenous PAs homeostasis to induce stress signal networks. In this report, the evolutionary study has been conducted to reveal the phylogenetic relationship of genes encoding enzymes of the anabolic and catabolic pathway of PAs among the five plant lineages including green algae, moss, lycophyte, dicot and monocot along with their respective exon–intron structural patterns. Our results indicated that natural selection pressure had considerable influence on the ancestral PA metabolic pathway coding genes of land plants. PA metabolic genes have undergone gradual evolution by duplication and diversification process leading to subsequent structural modification through exon–intron gain and loss events to acquire specific function under environmental stress conditions. We have illuminated on the potential regulation of both the pathways by investigating the real-time expression analyses of PA metabolic pathway related enzyme coding genes at the transcriptional level in root and shoot tissues of two indica rice varieties, namely IR 36 (salt sensitive) and Nonabokra (salt-tolerant) in response to salinity in presence or absence of exogenous spermidine (Spd) treatment. Additionally, we have performed tissue specific quantification of the intracellular PAs and tried to draw probable connection between the PA metabolic pathway activation and endogenous PAs accumulation. Our results successfully enlighten the fact that how exogenous Spd in presence or absence of salt stress adjust the intracellular PA pathways to equilibrate the cellular PAs that would have been attributed to plant salt tolerance.

Molecular and Functional Characterization of GR2-R1 Event Based Backcross Derived Lines of Golden Rice in the Genetic Background of a Mega Rice Variety Swarna

Homozygous Golden Rice lines developed in the background of Swarna through marker assisted backcross breeding (MABB) using transgenic GR2-R1 event as a donor for the provitamin A trait have high levels of provitamin A (up to 20 ppm) but are dwarf with pale green leaves and drastically reduced panicle size, grain number and yield as compared to the recurrent parent, Swarna. In this study, we carried out detailed morphological, biochemical and molecular characterization of these lines in a quest to identify the probable reasons for their abnormal phenotype. Nucleotide blast analysis with the primer sequences used to amplify the transgene revealed that the integration of transgene disrupted the native OsAux1 gene, which codes for an auxin transmembrane transporter protein. Real time expression analysis of the transgenes (ZmPsy and CrtI) driven by endosperm-specific promoter revealed the leaky expression of the transgene in the vegetative tissues. We propose that the disruption of OsAux1 disturbed the fine balance of plant growth regulators viz., auxins, gibberellic acid and abscisic acid, leading to the abnormalities in the growth and development of the lines homozygous for the transgene. The study demonstrates the conserved roles of OsAux1 gene in rice and Arabidopsis.

Mutation in LIM2 Is Responsible for Autosomal Recessive Congenital Cataracts.

PurposeTo identify the molecular basis of non-syndromic autosomal recessive congenital cataracts (arCC) in a consanguineous family.MethodsAll family members participating in the study received a comprehensive ophthalmic examination to determine their ocular phenotype and contributed a blood sample, from which genomic DNA was extracted. Available medical records and interviews with the family were used to compile the medical history of the family. The symptomatic history of the individuals exhibiting cataracts was confirmed by slit-lamp biomicroscopy. A genome-wide linkage analysis was performed to localize the disease interval. The candidate gene, LIM2 (lens intrinsic membrane protein 2), was sequenced bi-directionally to identify the disease-causing mutation. The physical changes caused by the mutation were analyzed in silico through homology modeling, mutation and bioinformatic algorithms, and evolutionary conservation databases. The physiological importance of LIM2 to ocular development was assessed in vivo by real-time expression analysis of Lim2 in a mouse model.ResultsOphthalmic examination confirmed the diagnosis of nuclear cataracts in the affected members of the family; the inheritance pattern and cataract development in early infancy indicated arCC. Genome-wide linkage analysis localized the critical interval to chromosome 19q with a two-point logarithm of odds (LOD) score of 3.25. Bidirectional sequencing identified a novel missense mutation, c.233G>A (p.G78D) in LIM2. This mutation segregated with the disease phenotype and was absent in 192 ethnically matched control chromosomes. In silico analysis predicted lower hydropathicity and hydrophobicity but higher polarity of the mutant LIM2-encoded protein (MP19) compared to the wild-type. Moreover, these analyses predicted that the mutation would disrupt the secondary structure of a transmembrane domain of MP19. The expression of Lim2, which was detected in the mouse lens as early as embryonic day 15 (E15) increased after birth to a level that was sustained through the postnatal time points.ConclusionA novel missense mutation in LIM2 is responsible for autosomal recessive congenital cataracts.

A Systematic View of the MLO Family in Rice Suggests Their Novel Roles in Morphological Development, Diurnal Responses, the Light-Signaling Pathway, and Various Stress Responses.

The Mildew resistance Locus O (MLO) family is unique to plants, containing genes that were initially identified as a susceptibility factor to powdery mildew pathogens. However, little is known about the roles and functional diversity of this family in rice, a model crop plant. The rice genome has 12 potential MLO family members. To achieve systematic functional assignments, we performed a phylogenomic analysis by integrating meta-expression data obtained from public sources of microarray data and real-time expression data into a phylogenic tree. Subsequently, we identified 12 MLO genes with various tissue-preferred patterns, including leaf, root, pollen, and ubiquitous expression. This suggested their functional diversity for morphological agronomic traits. We also used these integrated transcriptome data within a phylogenetic context to estimate the functional redundancy or specificity among OsMLO family members. Here, OsMLO12 showed preferential expression in mature pollen; OsMLO4, in the root tips; OsMLO10, throughout the roots except at the tips; and OsMLO8, expression preferential to the leaves and trinucleate pollen. Of particular interest to us was the diurnal expression of OsMLO1, OsMLO3, and OsMLO8, which indicated that they are potentially significant in responses to environmental changes. In osdxr mutants that show defects in the light response, OsMLO1, OsMLO3, OsMLO8, and four calmodulin genes were down-regulated. This finding provides insight into the novel functions of MLO proteins associated with the light-responsive methylerythritol 4-phosphate pathway. In addition, abiotic stress meta-expression data and real-time expression analysis implied that four and five MLO genes in rice are associated with responses to heat and cold stress, respectively. Upregulation of OsMLO3 by Magnaporthe oryzae infection further suggested that this gene participates in the response to pathogens. Our analysis has produced fundamental information that will enhance future studies of the diverse developmental or physiological phenomena mediated by the MLO family in this model plant system.