Ramalingam Sathishkumar

Stress-Induced Accumulation of DcAOX1 and DcAOX2a Transcripts Coincides with Critical Time Point for Structural Biomass Prediction in Carrot Primary Cultures (Daucus carota L.).

Stress-adaptive cell plasticity in target tissues and cells for plant biomass growth is important for yield stability. In vitro systems with reproducible cell plasticity can help to identify relevant metabolic and molecular events during early cell reprogramming. In carrot, regulation of the central root meristem is a critical target for yield-determining secondary growth. Calorespirometry, a tool previously identified as promising for predictive growth phenotyping has been applied to measure the respiration rate in carrot meristem. In a carrot primary culture system (PCS), this tool allowed identifying an early peak related with structural biomass formation during lag phase of growth, around the 4th day of culture. In the present study, we report a dynamic and correlated expression of carrot AOX genes (DcAOX1 and DcAOX2a) during PCS lag phase and during exponential growth. Both genes showed an increase in transcript levels until 36 h after explant inoculation, and a subsequent down-regulation, before the initiation of exponential growth. In PCS growing at two different temperatures (21°C and 28°C), DcAOX1 was also found to be more expressed in the highest temperature. DcAOX genes’ were further explored in a plant pot experiment in response to chilling, which confirmed the early AOX transcript increase prior to the induction of a specific anti-freezing gene. Our findings point to DcAOX1 and DcAOX2a as being reasonable candidates for functional marker development related to early cell reprogramming. While the genomic sequence of DcAOX2a was previously described, we characterize here the complete genomic sequence of DcAOX1.

Epigenetic silencing in transgenic plants

Epigenetic silencing is a natural phenomenon in which the expression of genes is regulated through modifications of DNA, RNA, or histone proteins. It is a mechanism for defending host genomes against the effects of transposable elements and viral infection, and acts as a modulator of expression of duplicated gene family members and as a silencer of transgenes. A major breakthrough in understanding the mechanism of epigenetic silencing was the discovery of silencing in transgenic tobacco plants due to the interaction between two homologous promoters. The molecular mechanism of epigenetic mechanism is highly complicated and it is not completely understood yet. Two different molecular routes have been proposed for this, that is, transcriptional gene silencing, which is associated with heavy methylation of promoter regions and blocks the transcription of transgenes, and post-transcriptional gene silencing (PTGS), the basic mechanism is degradation of the cytosolic mRNA of transgenes or endogenous genes. Undesired transgene silencing is of major concern in the transgenic technologies used in crop improvement. A complete understanding of this phenomenon will be very useful for transgenic applications, where silencing of specific genes is required. The current status of epigenetic silencing in transgenic technology is discussed and summarized in this mini-review.

Anti-chikungunya activity of luteolin and apigenin rich fraction from Cynodon dactylon

OBJECTIVE To obtain luteolin and apigenin rich fraction from the ethanolic extract of Cynodon dactylon (L.) (C. dactylon) Pers and evaluate the fractions cytotoxicity and anti-Chikungunya potential using Vero cells. METHODS The ethanolic extract of C. dactylon was subjected to silica gel column chromatography to obtain anti-chikungunya virus (CHIKV) fraction. Reverse phase-HPLC and GC-MS studies were carried out to identify the major phytochemicals in the fraction using phytochemical standards. Cytotoxicity and the potential of the fraction against CHIKV were evaluated in vitro using Vero cells. Reduction in viral replication was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) after treating the viral infected Vero cells with the fraction. RESULTS Reverse Phase-HPLC and GC-MS studies confirmed the presence of flavonoids, luteolin and apigenin as major phytochemicals in the anti-CHIKV ethanolic fraction of C. dactylon. The fraction was found to exhibit potent viral inhibitory activity (about 98%) at the concentration of 50 µg/mL as observed by reduction in cytopathic effect, and the cytotoxic concentration of the fraction was found to be 250 µg/mL. RT-PCR analyses indicated that the reduction in viral mRNA synthesis in fraction treated infected cells was much higher than the viral infected control cells. CONCLUSIONS Luteolin and apigenin rich ethanolic fraction from C. dactylon can be utilized as a potential therapeutic agent against CHIKV infection as the fraction does not show cytotoxicity while inhibiting the virus.

Chikungunya infection: A potential re-emerging global threat

Infectious diseases are indeed a lifelong threat to everyone irrespective of age, sex, lifestyle and socio-economic status. The infectious diseases have persisted among the prominent causes of death globally. Recently, re-emergence of Chikungunya viral infection harmed many in Asian and African countries. Chikungunya was considered as a major threat in developing and under-developed countries; the recent epidemiological outbreak of Chikungunya in La Reunion urges the global researchers to develop effective vaccine against this viral disease. In this review, Chikungunya, pathogenesis and epidemiology were briefly described.

Isolation and characterization of cold inducible genes in carrot by suppression subtractive hybridization

AbstactDaucus carota is cultivated widely but grows best in cool climates. Suppression subtractive hybridization (SSH) is a PCR based method used to selectively amplify differentially expressed cDNAs and simultaneously suppress non-target cDNA. A subtraction forward library was constructed using RNA isolated from the leaves of unstressed and cold stressed carrot plants to determine the genes upregulated during cold stress. Out of the hundreds of clones obtained, sequences of 41 promising clones were submitted to the NCBI EST database. Sequence analyses revealed that these genes have significant roles in signal transduction, osmolyte synthesis and transport, regulation of transcription, translation and protein folding. Semiquantitative real-time polymerase chain reaction analysis (sqRT-PCR) of Dc cyclin, Dc WD and Dc profilin shows that the first two genes were upregulated while Dc profilin was constitutively expressed, but the analyses of the same with SSH, a much more sensitive technique showed an upregulation of all three genes.

Overexpression of homogentisate phytyltransferase (HPT) and tocopherol cyclase (TC) enhances α-tocopherol content in transgenic tobacco

Photosynthetic organisms synthesize the amphipathic antioxidants called tocopherols which are essential components of the human diet. To increase the α-tocopherol (vitamin E) content, Arabidopsis genes encoding homogentisate phytyltransferase (HPT) and tocopherol cyclase (TC) were constitutively expressed individually and in combination (HPT:TC) in tobacco plant by Agrobacterium mediated transformation. The transgene was confirmed by polymerase chain reaction (PCR), transgene expression was studied by reverse transcriptase (RT)-PCR, integration of the transgene in the plant genome was confirmed by Southern blot, and α-tocopherol content was quantified using high performance liquid chromatography (HPLC). The α-tocopherol content in transgenic tobacco plants expressing HPT, TC, and HPT:TC was increased by 5.4-, 4.0-, and 7.1-fold, respectively, when compared to the wild type (WT). These results indicate that, the HPT and TC activities are critical for enhancing the vitamin E content in tobacco plants.

Abiotic Stress-Induced Redox Changes and Programmed Cell Death in Plants—A Path to Survival or Death?

Programmed cell death (PCD) is an active process, which occurs during growth, development and in response to various adverse environmental factors. During the course of growth and development, plants are frequently exposed to various stresses such as salinity, temperatures, heavy metals, drought and biotic factors. Plants have also evolved strategies to overcome these adverse conditions. However, when the intensity of these detrimental factors is high, plant cells undergo a process called PCD, as a part of defense mechanism. PCD is a highly regulated process, in which specific targeted cells are damaged to ensure the survival of the organism. Thus, the process facilitates the removal of unwanted and damaged cells, thus maintaining cellular differentiation and tissue homeostasis. PCD also plays an important role in developmental processes, such as differentiation of tracheary elements, formation of glandular trichomes, abscission of floral organs and embryo formation, and hence, it is a vital process for normal growth and development of plants. Stress-induced PCD significantly determines the crop yield and productivity, and hence, it is significant to agriculture. In this chapter, molecular mechanisms involved in PCD related to abiotic stress involving mitochondria and plastid are discussed.

Small RNAs: Master Regulators of Epigenetic Silencing in Plants

From fairly simple beginnings, research on epigenetic silencing in plants has revealed a highly complex epigenetic pathway. In the last two decades, several interesting phenomena associated with epigenetic regulation in plants were dissected giving insights into the biological significance of epigenetic marks and the role it plays in an organism’s life cycle by controlling different physiological processes like plant development, morphogenesis, reproduction, and stress response. Epigenetics refers to either heritable or reversible genetic modifications in DNA or histone proteins that maintain the nucleosome structure in a dynamic manner or those mediated by small RNAs (sRNAs) that in turn modulate gene expression. Plants are equipped with intricate regulatory mechanism to elicit highly sequence-specific chromatin-based gene silencing. Diverse classes of RNAs like small interfering RNA (siRNA), microRNAs (miRNAs), and long noncoding RNAs (lnc RNAs) have emerged as key regulators of gene expression along with several accessory proteins. sRNAs are widespread in various eukaryotes and are specifically involved in the maintenance of chromatin modifications in plants. These sRNAs regulate gene expression in different ways including post-transcriptional gene silencing (PTGS) in cytosol by targeting complementary transcripts for degradation, thereby repressing protein synthesis. In nucleus, sRNAs are responsible for transcriptional gene silencing (TGS) by directing epigenetic modifications like cytosine or histone methylation to homologous regions of the genome. This chapter gives an overview of the role of small RNAs in PTGS and TGS.

In vitro asymbiotic seed germination, mycorrhization and seedling development of Acampae praemorsa (Roxb.) Blatt. & Mc Cann, a common south Indian orchid

Abstract Objective To develope a conservation strategies for orchid species. Methods Seeds of Acampae praemorsa (Roxb.) Blatt. Mc Cann ( A. praemorsa ) were obtained from mature pods collected from Velliangiri hills and germinated on Murashige and Skoog (MS) medium supplemented with various concentration of Benzylaminopurine (BAP). Results Maximum seed germination (85%) was observed on MS media supplemented with 2 mg/L BAP. Seed germination percentage increased with increasing concentrations of BAP (0.5 mg/L to 2 mg/L), but 3 mg/L of BAP inhibited seed germination. Variations observed were significantly ( P ex vitro conditions. Conclusions In present study all the mycorrhizal seedlings survived, because orchid mycorrhizal fungi enhance growth of orchid plantlets and present study gives an effective protocol for seed germination and plantlet regeneration from immature seeds which can be used for establishing A. praemorsa populations in Velliangiri Hills and elsewhere.

An immunoinformatics approach to define T cell epitopes from polyketide and non-ribosomal peptide synthesis proteins of Mycobacterium tuberculosis as potential vaccine candidates

The role of polyketide and non‐ribosomal proteins from the class of small molecule metabolism of Mycobacterium tuberculosis is well documented in envelope organization, virulence, and pathogenesis. Consequently, the identification of T cell epitopes from these proteins could serve to define potential antigens for the development of vaccines. Fourty‐one proteins from polyketide and non‐ribosomal peptide synthesis of small molecule metabolism proteins of M tuberculosis H37Rv were analyzed computationally for the presence of HLA class I binding nanomeric peptides. All possible overlapping nanomeric peptide sequences from 41 small molecule metabolic proteins were generated through in silico and analyzed for their ability to bind to 33 alleles belonging to A, B, and C loci of HLA class I molecule. Polyketide and non‐ribosomal protein analyses revealed that 20% of generated peptides were predicted to bind HLA with halftime of dissociation T1/2 ≥ 100 minutes, and 77% of them were mono‐allelic in their binding. The structural bases for recognition of nanomers by different HLA molecules were studied by structural modeling of HLA class I‐peptide complexes. Pathogen peptides that could mimic as self‐peptides or partially self‐peptides in the host were excluded using a comparative study with the human proteome; thus, subunit or DNA vaccines will have more chance of success.