De novo transcriptome profiling unveils the regulation of phenylpropanoid biosynthesis in unripe Piper nigrum berries.

BACKGROUND: Black pepper (Piper nigrum L.) is rich in bioactive compounds that make it an imperative constituent in traditional medicines. Although the unripe fruits have long been used in different Ayurvedic formulations, the mechanism of gene regulation resulting in the production of the bioactive compounds in black pepper is not much investigated. Exploring the regulatory factors favouring the production of bioactive compounds ultimately help to accumulate the medicinally important content of black pepper. The factors that enhance the biosynthesis of these compounds could be potential candidates for metabolic engineering strategies to obtain a high level production of significant biomolecules. RESULTS: Being a non-model plant, de novo sequencing technology was used to unravel comprehensive information about the genes and transcription factors that are expressed in mature unripe green berries of P. nigrum from which commercially available black pepper is prepared. In this study, the key gene regulations involved in the synthesis of bioactive principles in black pepper was brought out with a focus on the highly expressed phenylpropanoid pathway genes. Quantitative real-time PCR analysis of critical genes and transcription factors in the different developmental stages from bud to the mature green berries provides important information useful for choosing the developmental stage that would be best for the production of a particular bioactive compound. Comparison with a previous study has also been included to understand the relative position of the results obtained from this study. CONCLUSIONS: The current study uncovered significant information regarding the gene expression and regulation responsible for the bioactivity of black pepper. The key transcription factors and enzymes analyzed in this study are promising targets for achieving a high level production of significant biomolecules through metabolic engineering.

Harnessing the Natural Pool of Polyketide and Non-ribosomal Peptide Family: A Route Map towards Novel Drug Development.

The emergence of communicable and non-communicable diseases has posed a health challenge for millions of people worldwide and is a major threat to the economic and social development in the coming century. The occurrence of the recent pandemic, SARS-CoV-2, caused by lethal severe acute respiratory syndrome coronavirus 2, is one such example. Rapid research and development of drugs for the treatment and management of these diseases have become an incredibly challenging task for the pharmaceutical industry. Although, substantial attention has been paid to the discovery of therapeutic compounds from natural sources having significant medicinal potential, their synthesis has made a slow progress. Hence, the discovery of new targets by the application of the latest biotechnological and synthetic biology approaches is very much the need of the hour. Polyketides (PKs) and non-ribosomal peptides (NRPs) found in bacteria, fungi and plants are a diverse family of natural products synthesized by two classes of enzymes: polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS). These enzymes possess immense biomedical potential due to their simple architecture, catalytic capacity, as well as diversity. With the advent of the latest in-silico and in-vitro strategies, these enzymes and their related metabolic pathways, if targeted, can contribute highly towards the biosynthesis of an array of potentially natural drug leads that have antagonist effects on biopolymers associated with various human diseases. In the face of the rising threat from multidrug-resistant pathogens, this will further open new avenues for the discovery of novel and improved drugs by combining natural and synthetic approaches. This review discusses the relevance of polyketides and non-ribosomal peptides and the improvement strategies for the development of their derivatives and scaffolds, and how they will be beneficial for future bioprospecting and drug discovery.

Non-Targeted Metabolite Profiling Reveals Host Metabolomic Reprogramming during the Interaction of Black Pepper with Phytophthora capsici.

Phytophthora capsici is one of the most destructive pathogens causing quick wilt (foot rot) disease in black pepper (Piper nigrum L.) to which no effective resistance has been defined. To better understand the P. nigrum-P. capsici pathosystem, we employed metabolomic approaches based on flow-infusion electrospray-high-resolution mass spectrometry. Changes in the leaf metabolome were assessed in infected and systemic tissues at 24 and 48 hpi. Principal Component Analysis of the derived data indicated that the infected leaves showed a rapid metabolic response by 24 hpi whereas the systemic leaves took 48 hpi to respond to the infection. The major sources of variations between infected leaf and systemic leaf were identified, and enrichment pathway analysis indicated, major shifts in amino acid, tricarboxylic acid cycle, nucleotide and vitamin B6 metabolism upon infection. Moreover, the individual metabolites involved in defensive phytohormone signalling were identified. RT-qPCR analysis of key salicylate and jasmonate biosynthetic genes indicated a transient reduction of expression at 24 hpi but this increased subsequently. Exogenous application of jasmonate and salicylate reduced P. capsici disease symptoms, but this effect was suppressed with the co-application of abscisic acid. The results are consistent with abscisic acid reprogramming, salicylate and jasmonate defences in infected leaves to facilitate the formation of disease. The augmentation of salicylate and jasmonate defences could represent an approach through which quick wilt disease could be controlled in black pepper.

Calcium-Dependent Protein Kinase in Ginger Binds with Importin-α through Its Junction Domain for Nuclear Localization, and Further Interacts with NAC Transcription Factor.

Calcium-dependent protein kinases (CDPKs) are important sensors of Ca2+ elevations in plant cells regulating the gene expression linked with various cellular processes like stress response, growth and development, metabolism, and cytoskeleton dynamics. Ginger is an extensively used spice due to its unique flavor and immense medicinal value. The two major threats that interfere with the large scale production of ginger are the salinity and drought stress. ZoCDPK1 (Zingiber officinale Calcium-dependent protein kinase 1) is a salinity and drought-inducible CDPK gene isolated from ginger and undergoes dynamic subcellular localization during stress conditions. ZoCDPK1, with signature features of a typical Ca2+ regulated kinase, also possesses a bipartite nuclear localization sequence (NLS) in its junction domain (JD). A striking feature in ZoCDPK1 is the rare occurrence of a coupling between the NLS in JD and consensus sequences in regulatory domain. Here, we further identified its nature of nuclear localization and its interaction partners. In the homology model generated for ZoCDPK1, the regulatory domain mimics the crystal structure of the regulatory domain in Arabidopsis CDPK1. Molecular docking simulation of importin (ZoIMPα), an important protein involved in nuclear translocation, into the NLS of ZoCDPK1 was well-visualized. Furthermore, the direct interaction of ZoCDPK1 and ZoIMPα proteins was studied by the yeast 2-hybrid (Y2H) system, which confirmed that junction domain (JD) is an important interaction module required for ZoCDPK1 and ZoIMPα binding. The probable interacting partners of ZoCDPK1 were also identified using Y2H experiment. Of the 10 different stress-related interacting partners identified for ZoCDPK1, NAC transcription factor (TF) needs special mention, especially in the context of ZoCDPK1 function. The interaction between ZoCDPK1 and NAC TF, in fact, corroborate with the results of gene expression and over-expression studies of ZoCDPK1. Hence ZoCDPK1 is operating through NAC TF mediated ABA-independent, cold non-responsive stress signaling pathway in ginger.

Unravelling the complexity of microRNA-mediated gene regulation in black pepper (Piper nigrum L.) using high-throughput small RNA profiling.

KEY MESSAGE: Analysis of high-throughput small RNA deep sequencing data, in combination with black pepper transcriptome sequences revealed microRNA-mediated gene regulation in black pepper ( Piper nigrum L.). Black pepper is an important spice crop and its berries are used worldwide as a natural food additive that contributes unique flavour to foods. In the present study to characterize microRNAs from black pepper, we generated a small RNA library from black pepper leaf and sequenced it by Illumina high-throughput sequencing technology. MicroRNAs belonging to a total of 303 conserved miRNA families were identified from the sRNAome data. Subsequent analysis from recently sequenced black pepper transcriptome confirmed precursor sequences of 50 conserved miRNAs and four potential novel miRNA candidates. Stem-loop qRT-PCR experiments demonstrated differential expression of eight conserved miRNAs in black pepper. Computational analysis of targets of the miRNAs showed 223 potential black pepper unigene targets that encode diverse transcription factors and enzymes involved in plant development, disease resistance, metabolic and signalling pathways. RLM-RACE experiments further mapped miRNA-mediated cleavage at five of the mRNA targets. In addition, miRNA isoforms corresponding to 18 miRNA families were also identified from black pepper. This study presents the first large-scale identification of microRNAs from black pepper and provides the foundation for the future studies of miRNA-mediated gene regulation of stress responses and diverse metabolic processes in black pepper.

Biodegradable tocopherol acetate as a drug carrier to prevent ureteral stent-associated infection.

Biomaterial-centred bacterial infections present common and challenging complications with medical implants like ureteral stent which provide substratum for the biofilm formation. Hence the purpose of this study is to make antibacterial stent surface with biodegradable polymer (tocopherol acetate) and anti-infective agents (norfloxacin and metronidazole) using a modified dip-coating procedure. This is done by impregnating the stent pieces in the anti-infective solution (a mixture of norfloxacin-metronidazole and polymer) for uniform surface coating (drug-carrier-coated stents). After coating, agar diffusion test was performed as qualitative test to find out the sensitivity of coated stents against the clinical isolates, Staphylococcus epidermidis and Escherichia coli. Quantitative test was measured by calculating the numbers of adhered bacteria on coated and uncoated stents by incubating the stent pieces in artificial urine. Difference in the number of viable bacteria adhered on the surface of coated and uncoated stents were statistically calculated using chi square test with p < 0.05 considered significant. The stent colonising ability of Staphylococcus epidermidis and Escherichia coli in a controlled environment chamber was determined using two-challenge dose of the isolates by in vitro challenge test. In qualitative test, the zone of inhibition around the coated stents showed sensitivity against the clinical isolates. In quantitative test, the number of adhered bacteria on the surface of coated stents was reduced to a significant level (p < 0.05). The polymer, tocopherol acetate is highly biodegradable in nature. Due to its degrading ability in body tissues, it releases the anti-infective drugs at a constant and sustained rate.