Muthu Thiruvengadam

The MADS box gene, FOREVER YOUNG FLOWER, acts as a repressor controlling floral organ senescence and abscission in Arabidopsis.

The ectopic expression of a MADS box gene FOREVER YOUNG FLOWER (FYF) caused a significant delay of senescence and a deficiency of abscission in flowers of transgenic Arabidopsis. The defect in floral abscission was found to be due to a deficiency in the timing of cell separation of the abscission zone cells. Down-regulation of INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) may contribute to the delay of the floral abscission in 35S:FYF flowers. FYF was found to be highly expressed in young flowers prior to pollination and was significantly decreased after pollination, a pattern that correlated with its function. Ethylene insensitivity in senescence/abscission and the down-regulation of ETHYLENE RESPONSE DNA-BINDING FACTOR 1 (EDF1) and EDF2, downstream genes in the ethylene response, in 35S:FYF Arabidopsis suggested a role for FYF in regulating senescence/abscission by suppressing the ethylene response. This role was further supported by the fact that 35S:FYF enhanced the delay of flower senescence/abscission in ethylene response 1 (etr1), ethylene-insensitive 2 (ein2) and constitutive triple response 1 (ctr1) mutants, which have defects in upstream genes of the ethylene signaling pathway. The presence of a repressor domain in the C-terminus of FYF and the enhancement of the delay of senescence/abscission in FYF+SRDX (containing a suppression motif) transgenic plants suggested that FYF acts as a repressor. Indeed, in FYF-DR+VP16 transgenic dominant-negative mutant plants, in which FYF was converted to a potent activator by fusion to a VP16-AD motif, the senescence/abscission of the flower organs was significantly promoted, and the expression of BOP2, IDA and EDF1/2 was up-regulated. Our data suggest a role for FYF in controlling floral senescence/abscission by repressing ethylene responses and regulating the expression of BOP2 and IDA in Arabidopsis.

Rheumatoid Arthritis: The Stride from Research to Clinical Practice.

Over 70 different genetic variants with a significant association with rheumatoid arthritis (RA) have been discovered. Anti-citrullination protein antibodies (ACPA)-positive RA variants are more well-defined than their ACPA-negative counterparts. The human leukocyte antigen, HLA-DRB1 locus remains the prime suspect in anti-citrullination protein antibodies (ACPA)—positive RA. Different HLA-DRB1 alleles are linked to RA susceptibility across different ethnicities. With evolving techniques, like genome-wide association studies (GWAS) and single nucleotide polymorphism (SNP) arrays, more non-HLA susceptibility loci have been identified for both types of RA. However, the functional significance of only a handful of these variants is known. Their roles include increasing susceptibility to RA or in determining the speed at which the disease progresses. Additionally, a couple of variations are associated with protection from RA. Defining such clear-cut biological functions can aid in the clinical diagnosis and treatment of RA. Recent research has focused on the implication of microRNAs, with miR-146a widely studied. In addition to disease susceptibility, genetic variations that influence the efficacy and toxicity of anti-RA agents have also been identified. Polymorphisms in the MTHFR gene influence the effectiveness of methotrexate, the first line of therapy in RA. Larger studies are, however, needed to identify potential biomarkers for early disease identification and monitoring disease progression.

Growth and Replication of Infectious Bursal Disease Virus in the DF-1 Cell Line and Chicken Embryo Fibroblasts

Infectious bursal disease virus (IBDV) causes a highly contagious disease in young chicks and leads to significant economic losses in the poultry industry. To determine a suitable cell line for IBDV infection, replication, and growth kinetics of the virus, DF-1 cells and chicken embryo fibroblasts (CEF) were used. The population doubling per day (Pd/D) was found to be higher in DF-1 as compared to CEF cells. A suitable time of infection (TOI) was established for increased production of virus and greater infectivity titers. The DF-1 and CEF cells were found to be susceptible to infection by producing marked cytopathic effects (CPEs), and the growth curves of IBDV in DF-1 and CEF cells were evaluated by infectivity assay using tissue culture infectious dose (TCID50). The cytopathic effects of the virus in DF-1 and CEF cells were found to be similar, but higher viral titers were detected in the DF-1 cells as compared to CEF. Thus the DF-1 cell line had a higher growth potential and infectivity, which will be of advantage in vaccine production.

Establishment of an efficient Agrobacterium tumefaciens -mediated leaf disc transformation of spine gourd ( Momordica dioica Roxb. ex Willd)

Spine gourd ( Momordica dioica Roxb. ex Willd) is a medicinally and economically important plant and also used as vegetable. In this study, we established an Agrobacterium tumefaciens -mediated transformation procedure for M. dioica . Leaf explants were incubated with A. tumefaciens strain LBA4404 containing a binary vector pBAL2 carrying the reporter gene β-glucuronidase intron (GUS-INT) and the marker gene neomycin phosphotransferase (NPTII). Following co-cultivation, leaf explants were cultured on Murashige and Skoog (MS) + Gamborg et al., (B 5 ) medium supplemented with 6.6 μM 2,4- dichlorophenoxyacetic acid (2,4-D) combined with 3.3 μM 6-benzylaminopurine (BAP) containing 100 mg L –1 kanamycin and 300 mg L -1 carbenicillin. Kanamycin-resistant calluses were induced from the leaf explants after three weeks. Shoot regeneration was achieved after transferring the calluses onto fresh selection medium 8.8 μM BAP and 2.2 μM 2,4-D. Transgenic shoots were excised from callus and elongated in MS medium fortified with 3.0 μM gibberellic acid (GA 3 ), 100 mg L -1 kanamycin and 300 mg L -1 carbenicillin. Finally, the shoots were rooted on MS basal medium supplemented with 3.0 μM indole 3-butyric acid (IBA) and 100 mg L -1 kanamycin. High transformation frequency was achieved by using three-day-old precultured leaf explants. Furthermore, the presence of acetosyringone (200 μM), infection of explants for 30 min and three days of cultivation proved to be critical factors for greatly improving the transformation efficiency. Incorporation and expression of the transgenes confirmed by polymerase chain reaction (PCR), Southern blot analysis, reverse transcription (RT)-PCR and GUS histochemical assay. Using this protocol, transgenic M. dioica plants can be obtained in approximately three months with a high transformation frequency of 9%. Key words : Agrobacterium tumefaciens, acetosyringone, growth regulators, GUS, genetic transformation, Momordica dioica .

Enhanced Production of Anthraquinones and Phenolic Compounds and Biological Activities in the Cell Suspension Cultures of Polygonum multiflorum

Anthraquinones (AQs) and phenolic compounds are important phytochemicals that are biosynthesized in cell suspension cultures of Polygonum multiflorum. We wanted to optimize the effects of plant growth regulators (PGRs), media, sucrose, l-glutamine, jasmonic acid (JA), and salicylic acid (SA) for the production of phytochemicals and biomass accumulation in a cell suspension culture of P. multiflorum. The medium containing Murashige and Skoog (MS) salts and 4% sucrose supplemented with 1 mg/L 2,4-dichlorophenoxyacetic acid, 0.5 mg/L thidiazuron, and 100 µM l-glutamine at 28 days of cell suspension culture was suitable for biomass accumulation and AQ production. Maximum biomass accumulation (12.5 and 12.35 g fresh mass (FM); 3 and 2.93 g dry mass (DM)) and AQ production (emodin 295.20 and 282 mg/g DM; physcion 421.55 and 410.25 mg/g DM) were observed using 100 µM JA and SA, respectively. JA- and SA-elicited cell cultures showed several-fold higher biomass accumulation and AQ production than the control cell cultures. Furthermore, the cell suspension cultures effectively produced 23 phenolic compounds, such as flavonols and hydroxycinnamic and hydroxybenzoic acid derivatives. PGR-, JA-, and SA-elicited cell cultures produced a higher amount of AQs and phenolic compounds. Because of these metabolic changes, the antioxidant, antimicrobial, and anticancer activities were high in the PGR-, JA-, and SA-elicited cell cultures. The results showed that the elicitors (JA and SA) induced the enhancement of biomass accumulation and phytochemical (AQs and phenolic compounds) production as well as biological activities in the cell suspension cultures of P. multiflorum. This optimized protocol can be developed for large-scale biomass accumulation and production of phytochemicals (AQs and phenolic compounds) from cell suspension cultures, and the phytochemicals can be used for various biological activities.

Making Sense of the Tangle: Insights into Chromatin Folding and Gene Regulation

Proximity ligation assays such as circularized chromosome conformation capture and high-throughput chromosome capture assays have shed light on the structural organization of the interphase genome. Functional topologically associating domains (TADs) that constitute the building blocks of genomic organization are disrupted and reconstructed during the cell cycle. Epigenetic memory, as well as the sequence of chromosomes, regulate TAD reconstitution. Sub-TAD domains that are invariant across cell types have been identified, and contacts between these domains, rather than looping, are speculated to drive chromatin folding. Replication domains are established simultaneously with TADs during the cell cycle and the two correlate well in terms of characteristic features, such as lamin association and histone modifications. CCCTC-binding factor (CTCF) and cohesin cooperate across different cell types to regulate genes and genome organization. CTCF elements that demarcate TAD boundaries are commonly disrupted in cancer and promote oncogene activation. Chromatin looping facilitates interactions between distant promoters and enhancers, and the resulting enhanceosome complex promotes gene expression. Deciphering the chromatin tangle requires comprehensive integrative analyses of DNA- and protein-dependent factors that regulate genomic organization.

In vitro regeneration from internodal explants of bitter melon ( Momordica charantia L.) via indirect organogenesis

Organogenic callus induction and high frequency shoot regeneration were achieved from internodal explants of bitter melon. About 97.5% of internodal explants derived from 30 day old in vivo grown plants produced green, compact nodular organogenic callus in Murashige and Skoog (MS) plus Gamborg et al. (1968) (B 5 ) medium containing 5.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.0 μM thidiazuron (TDZ) after two successive transfers at 11 days interval. Adventitious shoots were produced from organogenic callus when it was transferred to MS medium supplemented with 4.0 μM TDZ, 1.5 μM 2,4-D and 0.07 mM L-glutamine with shoot induction frequency of 96.5% and regeneration of adventitious shoots from callus (48 shoots per explant). Shoot proliferation occurred when callus with emerging shoots was transferred in the same medium at an interval of 15 days. The regenerated shoots were elongated on the same medium. The elongated shoots were rooted in MS medium supplemented with 3.0 μM indole 3-butyric acid (IBA). Rooted plants were acclimatized in green-house and subsequently established in soil with a survival rate of 95%. This protocol yielded an average of 48 shoots per internodal explant after 80 days of culture. Keywords: Adventitious shoots, growth regulators, hardening, organogenic callus, Momordica charantia

Jasmonic and salicylic acids enhanced phytochemical production and biological activities in cell suspension cultures of spine gourd (Momordica dioica Roxb)

In vitro cell suspension culture was established for the production of commercially valuable phytochemicals in Momordica dioica. The influence of elicitors in jasmonic acid (JA) and salicylic acid (SA) increased their effect on phytochemical production and biomass accumulation in M. dioica. The results indicate that compared with non-elicited cultures, JA- and SA-elicited cell suspension cultures had significantly enhanced phenolic, flavonoid, and carotenoid production, as well as antioxidant, antimicrobial, and antiproliferative activities. Furthermore, elicited cultures produced 22 phenolic compounds, such as flavonols, hydroxycinnamic acids, and hydroxybenzoic acids. Greater biomass production, phytochemical accumulation, and biological activity occurred in JA- than in SA-elicited cell cultures. This study is the first to successfully establish M. dioica cell suspension cultures for the production of phenolic compounds and carotenoids, as well as for biomass accumulation.

Expression of An Antisense Brassica oleracea GIGANTEA ( BoGI ) Gene in Transgenic Broccoli Causes Delayed Flowering, Leaf Senescence, and Post-Harvest Yellowing Retardation

Broccoli (Brassica oleracea L. var. italica) is an important vegetable crop all over the world. However, rapid post-harvest senescence in harvested floral heads reduces its value. Mutation in GIGANTEA (GI) caused delay of flowering and increased tolerance level to H2O2-induced oxidative stress in Arabidopsis. BoGI, a GI orthologue, was isolated and characterized from B. oleracea. BoGI mRNA is expressed throughout development and can be detected in leaves, stem, root, and flowers. Further analysis indicated that the expression of BoGI is modulated by the circadian clock. To investigate the senescence flowering-associated mechanism regulated by BoGI gene and the agricultural application of BoGI in controlling flowering time and floret yellowing for B. oleracea, constructs containing antisense cDNA of BoGI driven by 35S or a flower-specific AP1 promoter were transformed into B. oleracea and the transgenic plants were generated. The flowering time and the senescence of the detached leaves were significantly delayed in transgenic 35S::BoGI antisense plants. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed that clear reduction of BoGI expression was observed in these 35S::BoGI antisense plants compared to that in wild-type plants. Furthermore, post-harvest yellow and flower senescence was delayed in AP1::BoGI antisense plants. These findings indicate that BoGI could be involved in regulation of flowering time, leaf, floret, and flower senescence in broccoli.

Evaluation of polyphenol composition and biological activities of two samples from summer and winter seasons of Ligularia fischeri var. Spiciformis Nakai.

Phenolic contents, antioxidant and antimicrobial activities were determined by two samples from summer (June) and winter (December) seasons of Ligularia fischeri var. spiciformis Nakai. A total of 24 phenolic compounds were identified by ultra-performance liquid chromatography (UPLC) analysis. Myricetin (1964.35 and 1829.12 μg/g) was the most dominant flavonol compared to quercetin and kaempferol. Salicylic acid (222.80 and 215.25 μg/g) was the most important phenolic compound compared to pyrogallol, caffeic acid, gentisic acid, o-coumaric acid, gallic acid, protocatechuic acid and ferulic acid in summer (June) and winter (December) seasons. Phenolic contents and antioxidant capacities were estimated for the various solvent extracts (petroleum ether, butanol, ethyl acetate, methanol and water). Ethyl acetate extract exhibited the highest phenolic (332.64 and 299.44 mg/g gallic acid equivalent) and flavonoid contents (5.72 and 5.29 mg/g quercetin equivalent) and also the strongest antioxidant activity in summer and winter seasons. Due to these metabolic variations, the antioxidant and antimicrobial activities were increased with summer seasons compared to winter seasons. Our study shows that the samples collected in June had higher phenolic compounds, stronger antioxidative and antimicrobial activity than the samples of L. fischeri leaf extracts collected in December.