Embryo sac cellularization defects lead to supernumerary egg cells and twin embryos in Arabidopsis thaliana.

Arabidopsis lines with loss-of-function mutation in Embryo sac-specific Pectin MethylEsterase Inhibitor (Atepmei) gene showed seed sterility with embryo sac cellularization defects. Examination of tissue-cleared mature ovules revealed irregularly positioned nuclei/embryos within the embryo sacs. Egg cell-specific marker (DD45) expression analysis confirmed the presence of multiple egg cells in the mutant embryo sacs. These supernumerary egg cells were functional as evident from the production of twin embryos when supernumerary sperm cells were provided. The results of ruthenium red and tannic acid-ferric chloride staining of developing Atepmei mutant ovules showed that cell wall formation and maintenance were altered around embryo sac nuclei, which also coincided with change in the gamete specification. This report implicates the role of cell walls in gamete cell fate determination by altering cell-cell communication. Our analysis of the twin-embryo phenotype of epmei mutants also sheds light on the boundary conditions for double fertilization in plant reproduction.

Host-mediated RNAi of a Notch-like receptor gene in Meloidogyne incognita induces nematode resistance.

GLP-1 (abnormal germline proliferation) is a Notch-like receptor protein that plays an essential role in pharyngeal development. In this study, an orthologue of Caenorhabditis elegans glp-1 was identified in Meloidogyne incognita. A computational analysis revealed that the orthologue contained almost all the domains present in the C. elegans gene: specifically, the LIN-12/Notch repeat, the ankyrin repeat, a transmembrane domain and different ligand-binding motifs were present in orthologue, but the epidermal growth factor-like motif was not observed. An expression analysis showed differential expression of glp-1 throughout the life cycle of M. incognita, with relatively higher expression in the egg stage. To evaluate the silencing efficacy of Mi-glp-1, transgenic Arabidopsis plants carrying double-stranded RNA constructs of glp-1 were generated, and infection of these plants with M. incognita resulted in a 47-50% reduction in the numbers of galls, females and egg masses. Females obtained from the transgenic RNAi lines exhibited 40-60% reductions in the transcript levels of the targeted glp-1 gene compared with females isolated from the control plants. Second-generation juveniles (J2s), which were descendants of the infected females from the transgenic lines, showed aberrant phenotypes. These J2s exhibited a significant decrease in the overall distance from the stylet to the metacorpus region, and this effect was accompanied by disruption around the metacorporeal bulb of the pharynx. The present study suggests a role for this gene in organ (pharynx) development during embryogenesis in M. incognita and its potential use as a target in the management of nematode infestations in plants.

A part of the upstream promoter region of SHN2 gene directs trichome specific expression in Arabidopsis thaliana and heterologous plants.

A promoter trap mutant line of Arabidopsis carrying a promoterless ß-glucuronidase (uidA) gene exhibited GUS expression predominantly in all the trichomes. In this mutant, the T-DNA insertion was localized at 147bp upstream of the putative start codon, ATG, of the At5g11190 (SHN2) gene. Transcript profiling of the SHN2 suggested a constitutive expression of the gene in all the tissues. Deletion analysis of the upstream sequences established that a 565bp (-594/-30) region confers trichome-specific gene expression. The trichomes isolated from young, mature and senesced leaf tissues also showed the presence of SHN2 transcript. The occurrence of multiple TSSs on the SHN2 gene sequence, presence of the SHN2 transcript in the homozygous trip mutant, despite an insertional mutation event, and diverse reporter gene expression pattern driven by 5' and 3' promoter deletion fragments, suggest a complex transcriptional regulation of SHN2 gene in Arabidopsis. The promoter sequence -594/-30 showed a conserved functional role in conferring non-glandular trichome-specific expression in other heterologous systems like Brassica juncea and Solanum lycopersicon. Thus, in the present study T-DNA tagging has led to the identification of a trichome-specific regulatory sequence in the upstream region of a constitutively expressed SHN2 gene. The study also suggests a complex regulation of SHN2 gene. Isolated trichome specific region retains its functions in other systems like Brassica and tomato, hence could be effectively exploited in engineering trichome cells in heterologous crop plants to manipulate traits like biopharming and insect herbivory.

The TRAF Mediated Gametogenesis Progression (TRAMGaP) Gene Is Required for Megaspore Mother Cell Specification and Gametophyte Development.

In plants, the role of TRAF-like proteins with meprin and the TRAF homology (MATH) domain is far from clear. In animals, these proteins serve as adapter molecules to mediate signal transduction from Tumor Necrosis Factor Receptor to downstream effector molecules. A seed-sterile mutant with a disrupted TRAF-like gene (At5g26290) exhibiting aberrant gametogenesis led us to investigate the developmental role of this gene in Arabidopsis (Arabidopsis thaliana). The mutation was semidominant and resulted in pleiotropic phenotypes with such features as short siliques with fewer ovules, pollen and seed sterility, altered Megaspore Mother Cell (MMC) specification, and delayed programmed cell death in megaspores and the tapetum, features that overlapped those in other well-characterized mutants. Seed sterility and reduced transmission frequency of the mutant alleles pointed to a dual role, sporophytic and gametophytic, for the gene on the male side. The mutant also showed altered expression of various genes involved in such cellular and developmental pathways as regulation of transcription, biosynthesis and transport of lipids, hormone-mediated signaling, and gametophyte development. The diverse phenotypes of the mutant and the altered expression of key genes related to gametophyte and seed development could be explained based on the functional similarly between At5g26290 and MATH-BTB domain proteins that modulate gene expression through the ubiquitin-mediated proteasome system. These results show a novel link between a TRAF-like gene and reproductive development in plants.

Gametophyte Development Needs Mitochondrial Coproporphyrinogen III Oxidase Function.

Tetrapyrrole biosynthesis is one of the most essential metabolic pathways in almost all organisms. Coproporphyrinogen III oxidase (CPO) catalyzes the conversion of coproporphyrinogen III into protoporphyrinogen IX in this pathway. Here, we report that mutation in the Arabidopsis (Arabidopsis thaliana) CPO-coding gene At5g63290 (AtHEMN1) adversely affects silique length, ovule number, and seed set. Athemn1 mutant alleles were transmitted via both male and female gametes, but homozygous mutants were never recovered. Plants carrying Athemn1 mutant alleles showed defects in gametophyte development, including nonviable pollen and embryo sacs with unfused polar nuclei. Improper differentiation of the central cell led to defects in endosperm development. Consequently, embryo development was arrested at the globular stage. The mutant phenotype was completely rescued by transgenic expression of AtHEMN1 Promoter and transcript analyses indicated that AtHEMN1 is expressed mainly in floral tissues and developing seeds. AtHEMN1-green fluorescent protein fusion protein was found targeted to mitochondria. Loss of AtHEMN1 function increased coproporphyrinogen III level and reduced protoporphyrinogen IX level, suggesting the impairment of tetrapyrrole biosynthesis. Blockage of tetrapyrrole biosynthesis in the AtHEMN1 mutant led to increased reactive oxygen species (ROS) accumulation in anthers and embryo sacs, as evidenced by nitroblue tetrazolium staining. Our results suggest that the accumulated ROS disrupts mitochondrial function by altering their membrane polarity in floral tissues. This study highlights the role of mitochondrial ROS homeostasis in gametophyte and seed development and sheds new light on tetrapyrrole/heme biosynthesis in plant mitochondria.

Promoter Trapping and Deletion Analysis Show Arabidopsis thaliana APETALA2 Gene Promoter Is Bidirectional and Functions as a Pollen- and Ovule-Specific Promoter in the Reverse Orientation.

The Arabidopsis thaliana promoter trap mutant Bitrap-112 expressing green fluorescent protein (GFP) gene in the ovules was found to carry transferred DNA (T-DNA) insertion at -309 position of the APETALA2 (AP2) gene. Bitrap-112 line did not show phenotype associated with the AP2 mutation, suggesting that T-DNA insertion did not interrupt the AP2 promoter. Further, head-to-head orientation of GFP and AP2 genes indicated that the AP2 promoter could be bidirectional. A detailed deletion analysis of the upstream sequences of the AP2 gene was done to identify the promoter. GUS assay of transgenic A. thaliana plants carrying various AP2 upstream fragments fused to the uidA gene showed that ~200-bp 5' UTR sequences are capable of driving gene expression at low levels in vegetative tissues whereas inclusion of further upstream sequences (~300 bp) enhanced uidA expression comparable to native AP2 expression levels in various tissues including ovules. In the reverse orientation, the 519-bp AP2 upstream fragment was found to drive gene expression in immature ovules and pollen. Absence of antisense transcripts corresponding to the sequences upstream of AP2 gene in wild-type A. thaliana plants suggests that promoter trapping has uncovered a cryptic promoter, which in reverse orientation is capable of driving gene expression in ovules and anthers.

Identification, Validation and Utilization of Novel Nematode-Responsive Root-Specific Promoters in Arabidopsis for Inducing Host-Delivered RNAi Mediated Root-Knot Nematode Resistance.

The root-knot nematode (RKN), Meloidogyne incognita, is an obligate, sedentary endoparasite that infects a large number of crops and severely affects productivity. The commonly used nematode control strategies have their own limitations. Of late, RNA interference (RNAi) has become a popular approach for the development of nematode resistance in plants. Transgenic crops capable of expressing dsRNAs, specifically in roots for disrupting the parasitic process, offer an effective and efficient means of producing resistant crops. We identified nematode-responsive and root-specific (NRRS) promoters by using microarray data from the public domain and known conserved cis-elements. A set of 51 NRRS genes was identified which was narrowed down further on the basis of presence of cis-elements combined with minimal expression in the absence of nematode infection. The comparative analysis of promoters from the enriched NRRS set, along with earlier reported nematode-responsive genes, led to the identification of specific cis-elements. The promoters of two candidate genes were used to generate transgenic plants harboring promoter GUS constructs and tested in planta against nematodes. Both promoters showed preferential expression upon nematode infection, exclusively in the root in one and galls in the other. One of these NRRS promoters was used to drive the expression of splicing factor, a nematode-specific gene, for generating host-delivered RNAi-mediated nematode-resistant plants. Transgenic lines expressing dsRNA of splicing factor under the NRRS promoter exhibited upto a 32% reduction in number of galls compared to control plants.

The CarERF genes in chickpea (Cicer arietinum L.) and the identification of CarERF116 as abiotic stress responsive transcription factor.

The AP2/ERF family is one of the largest transcription factor gene families that are involved in various plant processes, especially in response to biotic and abiotic stresses. Complete genome sequences of one of the world's most important pulse crops chickpea (Cicer arietinum L.), has provided an important opportunity to identify and characterize genome-wide ERF genes. In this study, we identified 120 putative ERF genes from chickpea. The genomic organization of the chickpea ERF genes suggested that the gene family might have been expanded through the segmental duplications. The 120 member ERF family was classified into eleven distinct groups (I-X and VI-L). Transcriptional factor CarERF116, which is differentially expressed between drought tolerant and susceptible chickpea cultivar under terminal drought stress has been identified and functionally characterized. The CarERF116 encodes a putative protein of 241 amino acids and classified into group IX of ERF family. An in vitro CarERF116 protein-DNA binding assay demonstrated that CarERF116 protein specifically interacts with GCC box. We demonstrate that CarERF116 is capable of transactivation activity of and show that the functional transcriptional domain lies at the C-terminal region of the CarERF116. In transgenic Arabidopsis plants overexpressing CarERF116, significant up-regulation of several stress related genes were observed. These plants also exhibit resistance to osmotic stress and reduced sensitivity to ABA during seed germination. Based on these findings, we conclude that CarERF116 is an abiotic stress responsive gene, which plays an important role in stress tolerance. In addition, the present study leads to genome-wide identification and evolutionary analyses of chickpea ERF gene family, which will facilitate further research on this important group of genes and provides valuable resources for comparative genomics among the grain legumes.

Identification and characterization of Wilt and salt stress-responsive microRNAs in chickpea through high-throughput sequencing.

Chickpea (Cicer arietinum) is the second most widely grown legume worldwide and is the most important pulse crop in the Indian subcontinent. Chickpea productivity is adversely affected by a large number of biotic and abiotic stresses. MicroRNAs (miRNAs) have been implicated in the regulation of plant responses to several biotic and abiotic stresses. This study is the first attempt to identify chickpea miRNAs that are associated with biotic and abiotic stresses. The wilt infection that is caused by the fungus Fusarium oxysporum f.sp. ciceris is one of the major diseases severely affecting chickpea yields. Of late, increasing soil salinization has become a major problem in realizing these potential yields. Three chickpea libraries using fungal-infected, salt-treated and untreated seedlings were constructed and sequenced using next-generation sequencing technology. A total of 12,135,571 unique reads were obtained. In addition to 122 conserved miRNAs belonging to 25 different families, 59 novel miRNAs along with their star sequences were identified. Four legume-specific miRNAs, including miR5213, miR5232, miR2111 and miR2118, were found in all of the libraries. Poly(A)-based qRT-PCR (Quantitative real-time PCR) was used to validate eleven conserved and five novel miRNAs. miR530 was highly up regulated in response to fungal infection, which targets genes encoding zinc knuckle- and microtubule-associated proteins. Many miRNAs responded in a similar fashion under both biotic and abiotic stresses, indicating the existence of cross talk between the pathways that are involved in regulating these stresses. The potential target genes for the conserved and novel miRNAs were predicted based on sequence homologies. miR166 targets a HD-ZIPIII transcription factor and was validated by 5' RLM-RACE. This study has identified several conserved and novel miRNAs in the chickpea that are associated with gene regulation following exposure to wilt and salt stress.

Investigation of the Antioxidant and Hepatoprotective Potential of Hypericum mysorense.

BACKGROUND: Hypericum is a well-known plant genus in herbal medicine. Hypericum mysorense (Family: Hypericaceae), a plant belonging to the same genus, is well known in folklore medicine for its varied therapeutic potential. OBJECTIVE: The aim of the present study was to investigate the different parts of the plant for antioxidant and hepatoprotective properties. MATERIALS AND METHODS: The methanol extracts of Hypericum mysorense prepared from various parts of the plant were tested in vitro for their free radical scavenging activity against ABTS(•) (diammonium salt), DPPH(•) (1,1-diphenyl-2-picrylhydrazyl), NO(•), O2(•-) and (•)OH radicals, using standard systems of assays. The total antioxidant capacity, total phenolic and total flavonoid content of the extracts were analyzed. Further, the leaf and flowering top extracts were tested for their in vivo antioxidant and hepatoprotective activities on Wistar rats using a carbon tetrachloride-induced hepatic injury model. RESULTS: The leaf and flowering top extract showed potent antioxidant activity and also possessed highest total phenolic and flavonoid content. The antioxidant activity and the total phenolic and flavonoid content present in these extracts showed a good correlation. The leaf and flowering top extracts at 200 mg/kg restored aspartate amino transferase (ASAT), alanine amino transferase (ALAT), alkaline phosphatase (ALP), total bilirubin and protein levels significantly in CCl4-intoxicated rats. The tested extracts also showed a significant (p < 0.001) reduction in 2-thiobarbituric acid reactive substance (TBARS) levels with an increase in SOD and CAT levels. The histopathology of liver did not show any toxicity after the treatment with the extracts. The active extracts were standardized using two marker compounds, hyperoside and rutin, which were isolated from the plant by HPLC. HPLC studies revealed that the maximum concentration of hyperoside and rutin is present in the flowering top extract.

Can learning outcomes in cognitive domain be assessed effectively using multiple choice questions? A study in an undergraduate pharmacy curriculum

Background: The widespread use of multiple choice questions (MCQ) in examinations is attributed to its logistical advantage and broad coverage of content within a short duration. The end-of-semester examinations for several modules in the pharmacy programme previously employed a combination of written examination tools including MCQ, short answer questions (SAQ) or essays for assessing learning outcomes in the cognitive domain. Concerns regarding assessment fatigue and subjectivity in marking have led to a review of the assessment formats in the examinations. Various types of MCQ were consequently introduced as the only assessment tool. This study was conducted to evaluate the performance of students in the examinations as a result of the change. Methodology: Analyses were carried out on the end-ofsemester examination results of two cohorts of students for each module, one based on a combination of MCQ, SAQ or essay and the other based on MCQ alone. The class means were compared, and t-test was used to determine the difference between the performances. Results: Although the difference in the mean scores of the two groups is statistically significant in 13 of the 20 modules, the difference is less than 5% in 10 modules. Conclusion: The findings provide evidence that wellconstructed MCQ can effectively assess cognitive skills.

Characterization of a T-DNA promoter trap line of Arabidopsis thaliana uncovers a cryptic bi-directional promoter.

Investigation of the transgenic Arabidopsis promoter trap line GFP-868 that showed GFP expression only in anthers revealed the T-DNA insertion at 461bp upstream to the hypothetical gene At4g10596 with the GFP reporter gene in head-to-head orientation to the At4g10596 gene. The expression of the At4g10596 gene in wild type and in GFP-868 plant homozygous for T-DNA insertion was comparable and found in all tissues tested, while the GFP expression was restricted to anthers of the GFP-868 plants suggesting that the 461bp fragment separating the two genes in the GFP-868 line is functioning as bi-directional promoter. This 461bp fragment was cloned upstream to the GUS gene in two orientations to test for bi-directional promoter activity. Transgenic Arabidopsis plants carrying either of these constructs showed GUS activity in anthers indicating that this fragment behaves as bi-directional promoter specific to anthers. These results were also supported by the presence of cis-acting motifs such as TATA box and POLLEN1LELAT52 (AGAAA) within the 461bp sequence in both orientations. However, transcripts corresponding to the upstream sequences beyond -461 nucleotides were not detected in the wild type suggesting that this 461bp fragment is a cryptic promoter. The significance of the promoter trap approach and the usefulness of this type of promoter are discussed.

Molecular characterization of primary gene pool of chickpea based on ISSR markers.

Genetic diversity and relationships within and among members of the primary gene pool of chickpea, including 38 accessions of Cicer arietinum, six of C. reticulatum,, and four of C. echinospermum, were investigated using 31 ISSR markers. The study revealed moderate diversity, detecting 141 fragments, of which 79 (56%) were polymorphic. Averages were 0.125 for polymorphic information content, 0.350 for marker index, and 0.715 for resolving power. The UPGMA dendrogram and the principal coordinate analysis revealed a clear differentiation between wild and cultivated accessions. The clustering pattern did not strictly follow the grouping of accessions by geographic origin but was in good agreement with the pedigree data and the seed type. The study demonstrates that ISSRs provide promising marker tools in revealing genetic diversity and relationships in chickpea and can contribute to efficient identification, conservation, and utilization of germplasm for plant improvement through conventional as well as molecular breeding approaches.

Large-scale transcriptome analysis in chickpea (Cicer arietinum L.), an orphan legume crop of the semi-arid tropics of Asia and Africa.

Chickpea (Cicer arietinum L.) is an important legume crop in the semi-arid regions of Asia and Africa. Gains in crop productivity have been low however, particularly because of biotic and abiotic stresses. To help enhance crop productivity using molecular breeding techniques, next generation sequencing technologies such as Roche/454 and Illumina/Solexa were used to determine the sequence of most gene transcripts and to identify drought-responsive genes and gene-based molecular markers. A total of 103,215 tentative unique sequences (TUSs) have been produced from 435,018 Roche/454 reads and 21,491 Sanger expressed sequence tags (ESTs). Putative functions were determined for 49,437 (47.8%) of the TUSs, and gene ontology assignments were determined for 20,634 (41.7%) of the TUSs. Comparison of the chickpea TUSs with the Medicago truncatula genome assembly (Mt 3.5.1 build) resulted in 42,141 aligned TUSs with putative gene structures (including 39,281 predicted intron/splice junctions). Alignment of ∼37 million Illumina/Solexa tags generated from drought-challenged root tissues of two chickpea genotypes against the TUSs identified 44,639 differentially expressed TUSs. The TUSs were also used to identify a diverse set of markers, including 728 simple sequence repeats (SSRs), 495 single nucleotide polymorphisms (SNPs), 387 conserved orthologous sequence (COS) markers, and 2088 intron-spanning region (ISR) markers. This resource will be useful for basic and applied research for genome analysis and crop improvement in chickpea.

A comprehensive resource of drought- and salinity- responsive ESTs for gene discovery and marker development in chickpea (Cicer arietinum L.).

BACKGROUND: Chickpea (Cicer arietinum L.), an important grain legume crop of the world is seriously challenged by terminal drought and salinity stresses. However, very limited number of molecular markers and candidate genes are available for undertaking molecular breeding in chickpea to tackle these stresses. This study reports generation and analysis of comprehensive resource of drought- and salinity-responsive expressed sequence tags (ESTs) and gene-based markers. RESULTS: A total of 20,162 (18,435 high quality) drought- and salinity- responsive ESTs were generated from ten different root tissue cDNA libraries of chickpea. Sequence editing, clustering and assembly analysis resulted in 6,404 unigenes (1,590 contigs and 4,814 singletons). Functional annotation of unigenes based on BLASTX analysis showed that 46.3% (2,965) had significant similarity (< or =1E-05) to sequences in the non-redundant UniProt database. BLASTN analysis of unique sequences with ESTs of four legume species (Medicago, Lotus, soybean and groundnut) and three model plant species (rice, Arabidopsis and poplar) provided insights on conserved genes across legumes as well as novel transcripts for chickpea. Of 2,965 (46.3%) significant unigenes, only 2,071 (32.3%) unigenes could be functionally categorised according to Gene Ontology (GO) descriptions. A total of 2,029 sequences containing 3,728 simple sequence repeats (SSRs) were identified and 177 new EST-SSR markers were developed. Experimental validation of a set of 77 SSR markers on 24 genotypes revealed 230 alleles with an average of 4.6 alleles per marker and average polymorphism information content (PIC) value of 0.43. Besides SSR markers, 21,405 high confidence single nucleotide polymorphisms (SNPs) in 742 contigs (with > or = 5 ESTs) were also identified. Recognition sites for restriction enzymes were identified for 7,884 SNPs in 240 contigs. Hierarchical clustering of 105 selected contigs provided clues about stress- responsive candidate genes and their expression profile showed predominance in specific stress-challenged libraries. CONCLUSION: Generated set of chickpea ESTs serves as a resource of high quality transcripts for gene discovery and development of functional markers associated with abiotic stress tolerance that will be helpful to facilitate chickpea breeding. Mapping of gene-based markers in chickpea will also add more anchoring points to align genomes of chickpea and other legume species.

In Silico analysis of the lateral organ junction (LOJ) gene and promoter of Arabidopsis thaliana.

A T-DNA based promoter trapped mutant has led to the identification of a novel lateral organ junction specific promoter upstream of the pentatricopeptide repeat (PPR) protein coding gene LOJ in Arabidopsis thaliana by our laboratory. Various in silico based prediction tools are employed to characterize the upstream sequence of the LOJ gene. Out of numerous cis-elements detected in the LOJ promoter a few are considered important based on the expression pattern of the LOJ gene. These elements would provide a basis for designing experiments for more accurate promoter function annotation. A comparative search for conserved elements in the 5'-upstream region of a few genes involved in lateral organ development and meristem related expression reveals a few common relevant regulatory motifs. The coding region of the LOJ gene is intron-less and contains 19 PPR units. Based on in silico analysis, LOJ protein is predicted to be hydrophobic in nature and targeted to mitochondria. A partial 3D model of LOJ protein has been suggested using a homology-based modeling program.

T-DNA insertional mutagenesis in Arabidopsis: a tool for functional genomics

With the availability of complete genome sequences of several organisms, the focus has shifted from structural genomics to functional genomics, specifically in plants where the complete genomic sequences are becoming available i.e., Arabidopsis and rice. Agrobacterium mediated transformation which is exploited for transgenic technology is also being used as an effective mutagen and as a tool for functional genomics in higher plants. Besides the fact that the insertion of T-DNA element into a gene can lead to loss or gain of function, ingenious use of a variety of vectors have led to the identification of genes and regulatory elements in Arabidopsis. In this review, we highlight the progress made in the field of functional genomics of Arabidopsis using T-DNA tagging. Since this strategy has been very successfully employed in Arabidopsis and is now being extended to other plant species, we discuss the various vectors and experimental approaches employed to tag, identify and clone genes and promoter elements in Arabidopsis using T-DNA as a tool.