miRNA, phytometabolites and disease: Connecting the dots.

miRNAs are tiny noncoding ribonucleotides that function as critical regulators of gene-expression in eukaryotes. A single miRNA may be involved in the regulation of several target mRNAs forming complex cellular networks to regulate diverse aspects of development in an organism. The deregulation of miRNAs has been associated with several human diseases. Therefore, miRNA-based therapeutics is gaining interest in the pharmaceutical industry as the next-generation drugs for the cure of many diseases. Medicinal plants have also been used for the treatment of several human diseases and their curative potential is attributed to their reserve in bioactive metabolites. A role for miRNAs as regulators of the phytometabolic pathways in plants has emerged in the recent past. Experimental studies have also indicated the potential of plant encoded secondary phytometabolites to act as cross-regulators of mammalian miRNAs and transcripts to regulate human diseases (like cancer). The evidence for this cross-kingdom gene regulation through miRNA has gathered considerable enthusiasm in the scientific field, even though there are on-going debates regarding the reproducibility and the effectiveness of these findings. In this review, we provide information to connect the medicinal and gene regulatory properties of secondary phytometabolites, their regulation by miRNAs in plants and their effects on human miRNAs for regulating downstream metabolic or pathological processes. While further extensive research initiatives and good clinical evidence are required to prove or disapprove these findings, understanding of these regulations will have important implications in the potential use of synthetic or artificial miRNAs as effective alternatives for providing health benefits.

3Bs of CRISPR-Cas mediated genome editing in plants: exploring the basics, bioinformatics and biosafety landscape.

The recent thrust in research has projected the type II clustered regularly interspaced short palindromic repeats and associated protein 9 (CRISPR-Cas9) system as an avant-garde plant genome editing tool. It facilitates the induction of site-specific double-stranded DNA cleavage by the RNA-guided DNA endonuclease (RGEN), Cas9. Elimination, addition, or alteration of sections in DNA sequence besides the creation of a knockout genotype (CRISPRko) is aided by the CRISPR-Cas9 system in its wild form (wtCas9). The inactivation of the nuclease domain generates a dead Cas9 (dCas9), which is capable of targeting genomic DNA without scissoring it. The dCas9 system can be engineered by fusing it with different effectors to facilitate transcriptional activation (CRISPRa) and transcriptional interference (CRISPRi). CRISPR-Cas thus holds tremendous prospects as a genome-manipulating stratagem for a wide gamut of crops. In this article, we present a brief on the fundamentals and the general workflow of the CRISPR-Cas system followed by an overview of the prospects of bioinformatics in propelling CRISPR-Cas research with a special thrust on the available databases and algorithms/web-accessible applications that have aided in increasing the usage and efficiency of editing. The article also provides an update on the current regulatory landscape in different countries on the CRISPR-Cas edited plants to emphasize the far-reaching impact of the genomic editing technology. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01397-3.

Nanomaterials coupled with microRNAs for alleviating plant stress: a new opening towards sustainable agriculture.

Plant growth and development is influenced by their continuous interaction with the environment. Their cellular machinery is geared to make rapid changes for adjusting the morphology and physiology to withstand the stressful changes in their surroundings. The present scenario of climate change has however intensified the occurrence and duration of stress and this is getting reflected in terms of yield loss. A number of breeding and molecular strategies are being adopted to enhance the performance of plants under abiotic stress conditions. In this context, the use of nanomaterials is gaining momentum. Nanotechnology is a versatile field and its application has been demonstrated in almost all the existing fields of science. In the agriculture sector, the use of nanoparticles is still limited, even though it has been found to increase germination and growth, enhance physiological and biochemical activities and impact gene expression. In this review, we have summarized the use and role of nanomaterial and small non-coding RNAs in crop improvement while highlighting the potential of nanomaterial assisted eco-friendly delivery of small non-coding RNAs as an innovative strategy for mitigating the effect of abiotic stress.

Genomic properties of allamanda leaf mottle distortion virus, a new begomovirus from golden trumpet (Allamanda cathartica) in India.

Golden trumpet (Allamanda cathartica) plants were observed to exhibit mottling and distortion symptoms on leaves. The genome of an associated begomovirus (Al-K1) was amplified by rolling-circle amplification, cloned, and sequenced. The viral genome consisted of two circular ssDNA molecules, and the organization of the ORFs was similar to those of DNA-A and DNA-B components of bipartite begomoviruses. The size of DNA-A (KC202818) and DNA-B (MG969497) of the begomovirus was 2772 and 2690 nucleotides, respectively. Sequence analysis revealed that the DNA-A and DNA-B components shared the highest sequence identity with duranta leaf curl virus (MN537564, 87.8%) and cotton leaf curl Alabad virus (MH760452, 81.0%), respectively. Interestingly, the Al-K1 isolate shared significantly less nucleotide sequence identity with allamanda leaf curl virus (EF602306, 71.6%), the only monopartite begomovirus reported previously in golden trumpet from China. Al-K1 shared less than 91% sequence identity with other begomoviruses, and hence, according to the latest ICTV guidelines for species demarcation of begomoviruses, Al-K1 is proposed to be a member of a new species, and we propose the name "allamanda leaf mottle distortion virus" (AllLMoDV-[IN-Al_K1-12]) for this virus. AllLMoDV was detected in various golden trumpet samples from different locations by PCR with specific primers based on the genome sequence determined in this study. Our study provides evidence of the occurrence of a new bipartite begomovirus in a perennial ornamental plant in India.

Interaction between ßC1 of satellite and coat protein of Chili leaf curl virus plays a crucial role in suppression of host RNA silencing.

The monopartite Chili leaf curl virus (ChiLCV) and its ß-satellite (ChiLCB) have been found to co-exist in infected plants. The ability of ßC1 protein to suppress RNA silencing was investigated using an in-house developed in-planta reversal of silencing assay, using Nicotiana tabacum lines harboring green fluorescent protein (GFP) silenced by short hairpin GFP (ShGFP). Transient expression of recombinant ßC1 complemented and increased the suppressor activity of ChiLCV coat protein (CP), and this was confirmed by molecular analysis. In silico analysis followed by a yeast two-hybrid screen-identified ChiLCV-CP as the interacting partner of the ChiLCB-ßC1 protein. Subcellular localization through confocal analysis revealed that when ßC1 and ChiLCV-CP were co-present, the fluorescence was localized in the cytoplasm indicating that nuclear localization of both proteins was obstructed. The cytoplasmic compartmentalization of the two viral suppressors of RNA silencing may be responsible for the enhanced suppression of the host gene silencing. This study presents evidence on the interaction of ChiLCV-CP and ßC1 proteins and indicates that ChiLCB may support the ChiLCV in overcoming host gene silencing to cause Chili leaf curl disease. KEY POINTS: • CP of ChiLCV and ßC1 of ChiLCB contain RNA silencing suppression activity • The RNA silencing suppression activity of ChiLCB-ßC1 complements that of ChiLCV-CP • There is a direct interaction between ChiLCB-ßC1 and ChiLCV-CP.

Influence of virus-host interactions on plant response to abiotic stress.

Environmental factors play a significant role in controlling growth, development and defense responses of plants. Changes in the abiotic environment not only significantly alter the physiological and molecular pathways in plants, but also result in attracting the insect pests that carry a payload of viruses. Invasion of plants by viruses triggers the RNA silencing based defense mechanism in plants. In counter defense the viruses have gained the ability to suppress the host RNA silencing activities. A new paradigm has emerged, with the recognition that plant viruses also have the intrinsic capacity to modulate host plant response to environmental cues, in an attempt to favour their own survival. Thus, plant-virus interactions provide an excellent system to understand the signals in crosstalk between biotic (virus) and abiotic stresses. In this review, we have summarized the basal plant defense responses to pathogen invasion while emphasizing on the role of RNA silencing as a front line of defense response to virus infection. The emerging knowledge indicates overlap between RNA silencing with the innate immune responses during antiviral defense. The suppressors of RNA silencing serve as Avr proteins, which can be recognized by the host R proteins. The defense signals also function in concert with the phytohormones to influence plant responses to abiotic stresses. The current evidence on the role of virus induced host tolerance to abiotic stresses is also discussed.

Non-Coding RNAs in Response to Drought Stress.

Drought stress causes changes in the morphological, physiological, biochemical and molecular characteristics of plants. The response to drought in different plants may vary from avoidance, tolerance and escape to recovery from stress. This response is genetically programmed and regulated in a very complex yet synchronized manner. The crucial genetic regulations mediated by non-coding RNAs (ncRNAs) have emerged as game-changers in modulating the plant responses to drought and other abiotic stresses. The ncRNAs interact with their targets to form potentially subtle regulatory networks that control multiple genes to determine the overall response of plants. Many long and small drought-responsive ncRNAs have been identified and characterized in different plant varieties. The miRNA-based research is better documented, while lncRNA and transposon-derived RNAs are relatively new, and their cellular role is beginning to be understood. In this review, we have compiled the information on the categorization of non-coding RNAs based on their biogenesis and function. We also discuss the available literature on the role of long and small non-coding RNAs in mitigating drought stress in plants.

Serotonin and Melatonin Biosynthesis in Plants: Genome-Wide Identification of the Genes and Their Expression Reveal a Conserved Role in Stress and Development.

Serotonin (Ser) and melatonin (Mel) serve as master regulators of plant growth and development by influencing diverse cellular processes. The enzymes namely, tryptophan decarboxylase (TDC) and tryptamine 5-hydroxylase (T5H) catalyse the formation of Ser from tryptophan. Subsequently, serotonin N-acetyl transferase (SNAT) and acetyl-serotonin methyltransferase (ASMT) form Mel from Ser. Plant genomes harbour multiple genes for each of these four enzymes, all of which have not been identified. Therefore, to delineate information regarding these four gene families, we carried out a genome-wide analysis of the genes involved in Ser and Mel biosynthesis in Arabidopsis, tomato, rice and sorghum. Phylogenetic analysis unravelled distinct evolutionary relationships among these genes from different plants. Interestingly, no gene family except ASMTs showed monocot- or dicot-specific clustering of respective proteins. Further, we observed tissue-specific, developmental and stress/hormone-mediated variations in the expression of the four gene families. The light/dark cycle also affected their expression in agreement with our quantitative reverse transcriptase-PCR (qRT-PCR) analysis. Importantly, we found that miRNAs (miR6249a and miR-1846e) regulated the expression of Ser and Mel biosynthesis under light and stress by influencing the expression of OsTDC5 and OsASMT18, respectively. Thus, this study may provide opportunities for functional characterization of suitable target genes of the Ser and Mel pathway to decipher their exact roles in plant physiology.

Identification of microRNA-target modules from rice variety Pusa Basmati-1 under high temperature and salt stress.

High temperature and salinity stress are major factors limiting the growth and productivity of rice crop on a global scale. It is therefore an essential prerequisite to understand the molecular genetic regulation of plant responses to dual stresses. MicroRNAs (miRs) are recognized as key controllers of gene expression which act mainly at the post-transcriptional level to regulate various aspects of plant development. The present study attempts to investigate the miR circuits that are modulated in response to high temperature and salinity stress in rice. To gain insights into the pathway, preliminary miR profiles were generated using the next-generation sequencing (NGS) datasets. The identified molecules were filtered on the basis of fold differential regulation under high temperature, and time kinetics of their expression under the two individual stresses was followed to capture the regulatory windows. The analysis revealed the involvement of common miR regulatory nodes in response to two different abiotic stresses, thereby broadening our perspective about the stress-mediated regulatory mechanisms operative in rice.

Genetic modification in Malaysia and India: current regulatory framework and the special case of non-transformative RNAi in agriculture.

Recent developments in modern biotechnology such as the use of RNA interference (RNAi) have broadened the scope of crop genetic modification. RNAi strategies have led to significant achievements in crop protection against biotic and abiotic stresses, modification of plant traits, and yield improvement. As RNAi-derived varieties of crops become more useful in the field, it is important to examine the capacity of current regulatory systems to deal with such varieties, and to determine if changes are needed to improve the existing frameworks. We review the biosafety frameworks from the perspective of developing countries that are increasingly involved in modern biotechnology research, including RNAi applications, and make some recommendations. Malaysia and India have approved laws regulating living modified organisms and products thereof, highlighting that the use of any genetically modified step requires regulatory scrutiny. In view of production methods for exogenously applied double-stranded RNAs and potential risks from the resulting double-stranded RNA-based products, we argue that a process-based system may be inappropriate for the non-transformative RNAi technology. We here propose that the current legislation needs rewording to take account of the non-transgenic RNAi technology, and discuss the best alternative for regulatory systems in India and Malaysia in comparison with the existing frameworks in other countries.

Mapping the microRNA Expression Profiles in Glyoxalase Over-expressing Salinity Tolerant Rice.

In the recent years, glyoxalase pathway has been an active area of research in both human and plants. This pathway is reported to confer stress tolerance in plants, by modulating the glutathione homeostasis to achieve detoxification of a potent cytotoxic and mutagenic compound, methylglyoxal. The microRNAs (miRNAs) are also reported to play significant role in stress tolerance for plants. However, the cross-talk of miRNAs with the metabolism regulated by glyoxalase in the salinity-tolerance is unexplored. We therefore investigated whether expression profiles of miRNAs are altered in response to glyoxalase overexpression, and if any of these are also responsible for modulating the stress responses of plants. In this study, the Next Generation Sequencing (NGS) was employed to profile miRNA expression levels from glyoxalase overexpressing transgenic lines. The associated targets of differentially expressed miRNAs were predicted and their functional annotation was carried out using Gene Ontology (GO) and KEGG Orthology (KO), which showed their involvement in several crucial biological pathways. The analysis of NGS datasets also identified other isoforms or isomiRs of selected miRNAs, which may have an active role in developing tolerance against salt stress. Different aspects of miRNA modifications were also studied in glyoxalase overexpressing lines.

Identification and comparative analysis of microRNAs from tomato varieties showing contrasting response to ToLCV infections.

Increasing incidence of viral infections in crop plants adversely affects their growth and yield. Tomato (Solanum lycopersicum) is considered to be a favorite host for viruses with over 50 species of begomoviruses naturally infecting this crop. Tomato leaf curl virus (ToLCV) is among the most widespread and devastating begomoviruses affecting tomato production. microRNAs (miRs) have been established as key regulators of gene expression and plant development. The miR pathways are disturbed during infection by viruses. Thus, comprehension of regulatory miR networks is crucial in understanding the effect of viral pathogenicity. To identify key miRs involved in ToLCV infection, a high throughput approach involving next generation sequencing was employed. Healthy and infected leaf tissues of two tomato varieties, differing in their susceptibility to ToLCV infection were analyzed. NGS data analysis followed by computational predictions, led to identification of 91 known miRs, 15 novel homologs and 53 novel miRs covering two different varieties of tomato, susceptible (Pusa Ruby) and tolerant (LA1777) to ToLCV infection. The cleaved targets of these miRs were identified using online available degradome libraries from leaf, flower and fruit of tomato and showed their involvement in various biological pathways through KEGG Orthology. With detailed comparative profiling of expression pattern of these miRs, we could associate the specific miRs with the resistant and infected genotypes. This study depicted that in depth analysis of miR expression patterns and their functions will help in identification of molecules that can be used for manipulation of gene expression to increase crop production and developing resistance against diseases.

Artificial miRNA-mediated down-regulation of two monolignoid biosynthetic genes (C3H and F5H) cause reduction in lignin content in jute.

Artificial microRNAs (amiRNA) provide a new feature in the gene silencing era. Concomitantly, reducing the amount of lignin in fiber-yielding plants such as jute holds significant commercial and environmental potential, since this amount is inversely proportional to the quality of the fiber. The present study aimed at reducing the lignin content in jute, by introducing amiRNA based vectors for down-regulation of two monolignoid biosynthetic genes of jute, coumarate 3-hydroxylase (C3H) and ferulate 5-hydroxylase (F5H). The transgenic lines of F5H-amiRNA and C3H-amiRNA showed a reduced level of gene expression, which resulted in about 25% reduction in acid insoluble lignin content for whole stem and 12-15% reduction in fiber lignin as compared to the non-transgenic plants. The results indicate successful F5H-amiRNA and C3H-amiRNA transgenesis for lignin reduction in jute. This is likely to have far-reaching commercial implications and economic acceleration for jute producing countries.

3' and 5' microRNA-end post-biogenesis modifications in plant transcriptomes: Evidences from small RNA next generation sequencing data analysis.

The processing of miRNA from its precursors is a precisely regulated process and after biogenesis, the miRNAs are amenable to different kinds of modifications by the addition or deletion of nucleotides at the terminal ends. However, the mechanism and functions of such modifications are not well studied in plants. In this study, we have specifically analysed the terminal end non-templated miRNA modifications, using NGS data of rice, tomato and Arabidopsis small RNA transcriptomes from different tissues and physiological conditions. Our analysis reveals template independent terminal end modifications in the mature as well as passenger strands of the miRNA duplex. Interestingly, it is also observed that miRNA sequences terminating with a cytosine (C) at the 3' end undergo a higher percentage of 5' end modifications. The terminal end modifications did not correlate with the miRNA abundances and are independent of tissue types, physiological conditions and plant species. Our analysis indicates that the addition of nucleotides at miRNA ends is not influenced by the absence of RNA dependent RNA polymerase 6. Moreover the terminal end modified miRNAs are also observed amongst AGO1 bound small RNAs and have potential to alter target, indicating its important functional role in repression of gene expression.

Molecular priming with H2O2 and proline triggers antioxidant enzyme signals in maize seedlings during drought stress.

BACKGROUND: Drought and water stress impose major limitations to crops, including Maize, as they affect the plant biology at multiple levels. Drought activates the cellular signalling machinery to maintain the osmotic and ROS homeostasis for controlling plant response and adaptation to stress. Molecular priming of seeds plays a significant role in imparting stress tolerance by helping plants to remember the stress, which improves their response when they encounter stress again. METHODS: In this study, we examined the effect of priming maize seeds with H2O2 and proline, individually or in combination, on response to drought stress. We investigated the role of molecular priming on the physiological, biochemical and molecular response of maize seedlings during drought stress. RESULTS: We observed that seed-priming played a significant role in mediating stress tolerance of seedlings under drought stress as indicated by changes in growth, biochemical properties, pigment and osmolyte accumulation, antioxidant enzyme activities, gas exchange parameters and gene expression. Seed-priming resulted in reduced expression of specific miRNAs to increase target transcripts associated with synthesis of osmolytes and maintenance of ROS homeostasis for reducing potential damage to the cellular components. CONCLUSIONS: Seed-priming induced changes in the growth, biochemical properties, pigment and osmolyte accumulation, antioxidant enzyme activities, gas exchange parameters and gene expression, though the response was dependent on the genotype, as well as concentration and combination of the priming agents.

miRNAs and genes as molecular regulators of rice grain morphology and yield.

Rice is one of the most consumed crops worldwide and the genetic and molecular basis of its grain yield attributes are well understood. Various studies have identified different yield-related parameters in rice that are regulated by the microRNAs (miRNAs). MiRNAs are endogenous small non-coding RNAs that silence gene expression during or after transcription. They control a variety of biological or genetic activities in plants including growth, development and response to stress. In this review, we have summarized the available information on the genetic control of panicle architecture and grain yield (number and morphology) in rice. The miRNA nodes that are associated with their regulation are also described while focussing on the central role of miR156-SPL node to highlight the co-regulation of two master regulators that determine the fate of panicle development. Since abiotic stresses are known to negatively affect yield, the impact of abiotic stress induced alterations on the levels of these miRNAs are also discussed to highlight the potential of miRNAs for regulating crop yields.

Micro-regulators of auxin action.

microRNAs (miRs) are 21- to 24-nucleotide-long RNA molecules that are mainly involved in regulating the gene expression at the post-transcriptional levels. They are present in a variety of organisms from algae to plants and play an important role in gene regulation. The identification of several diverging and converging functions of miRs indicates that they play versatile roles in regulating plant development including differentiation, organ development, phase change, signalling, disease resistance and response to environmental stresses. This article provides a concise update on the plant miR functions and their targets in the auxin pathway with focus on the interactions between miRs and auxin signalling to intricately regulate the plant responses.

Identification of mirtrons in rice using MirtronPred: a tool for predicting plant mirtrons.

Studies from flies and insects have reported the existence of a special class of miRNA, called mirtrons that are produced from spliced-out introns in a DROSHA-independent manner. The spliced-out lariat is debranched and refolded into a stem-loop structure resembling the pre-miRNA, which can then be processed by DICER into mature ~21 nt species. The mirtrons have not been reported from plants. In this study, we present MirtronPred, a web based server to predict mirtrons from intronic sequences. We have used the server to predict 70 mirtrons in rice introns that were put through a stringent selection filter to shortlist 16 best sequences. The prediction accuracy was subsequently validated by northern analysis and RT-PCR of a predicted Os-mirtron-109. The target sequences for this mirtron were also found in the rice degradome database. The possible role of the mirtron in rice regulon is discussed. The MirtronPred web server is available at http://bioinfo.icgeb.res.in/mirtronPred.

Accelerated Long-Fragment Circular PCR for Genetic Manipulation of Plant Viruses in Unveiling Functional Genomics.

Molecular cloning, a crucial prerequisite for engineering plasmid constructs intended for functional genomic studies, relies on successful restriction and ligation processes. However, the lack of unique restriction sites often hinders construct preparation, necessitating multiple modifications. Moreover, achieving the successful ligation of large plasmid constructs is frequently challenging. To address these limitations, we present a novel PCR strategy in this study, termed 'long-fragment circular-efficient PCR' (LC-PCR). This technique involves one or two rounds of PCR with an additional third-long primer that complements both ends of the newly synthesized strand of a plasmid construct. This results in self-circularization with a nick-gap in each newly formed strand. The LC-PCR technique was successfully employed to insert a partial sequence (210 nucleotides) of the phytoene desaturase gene from Nicotiana benthamiana and a full capsid protein gene (770 nucleotides) of a begomovirus (tomato leaf curl New Delhi virus) into a 16.4 kb infectious construct of a tobamovirus, cucumber green mottle mosaic virus (CGMMV), cloned in pCambia. This was done to develop the virus-induced gene silencing vector (VIGS) and an expression vector for a foreign protein in plants, respectively. Furthermore, the LC-PCR could be applied for the deletion of a large region (replicase enzyme) and the substitution of a single amino acid in the CGMMV genome. Various in planta assays of these constructs validate their biological functionality, highlighting the utility of the LC-PCR technique in deciphering plant-virus functional genomics. The LC-PCR is not only suitable for modifying plant viral genomes but also applicable to a wide range of plant, animal, and human gene engineering under in-vitro conditions. Additionally, the LC-PCR technique provides an alternative to expensive kits, enabling quick introduction of modifications in any part of the nucleotide within a couple of days. Thus, the LC-PCR proves to be a suitable 'all in one' technique for modifying large plasmid constructs through site-directed gene insertion, deletion, and mutation, eliminating the need for restriction and ligation.