Temporal dynamics of endophytic bacterial and fungal communities during spike development in Piper longum L.

The female spikes (fruits) of Piper longum are widely used in Ayurvedic, Siddha and Unani medicine systems to treat respiratory and digestive disorders. The spikes are rich in piperine, a pharmacologically active amide alkaloid and a potent bioavailability enhancer, which accumulates to the highest level during the dark-green stage of spike development. Plant-associated microbiota influence the plant's fitness, response, and production of economically important metabolites. Considering the economic importance of piperine and other spike-derived alkaloids, understanding microbial community dynamics during spike development would be key to bioprospecting for economically important metabolites. In the present study, the structural diversity of microbial communities associated with early (SI), mid (SII), and late (SIII) stages of spike development in P. longum has been analysed by Illumina-based amplicon sequencing of 16S rRNA gene and ITS region. Results revealed that spike development significantly drives the diversity and abundance of spike-associated microbiota, especially bacterial communities. Cyanobacteria and Ascomycota constituted the most abundant bacterial and fungal phyla, respectively, across all stages of spike development. Interestingly, Halomonas, Kushneria and Haererehalobacter were found to be exclusively associated with SIII (corresponding to economically important) stage of spike development. Sphingomonas, Mortierella, Cladosporium and Vishniacozyma constituted the core microbiome of the spike. Besides, PICRUSt analysis revealed that amino acid metabolism was the most dominant metabolic function attributed to spike-associated bacterial communities. To the best of our knowledge, this is the first study to investigate the endomicrobiome dynamics during spike development in a medicinal plant species. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01352-2.

CRY2 gene of rice (Oryza sativa subsp. indica) encodes a blue light sensory receptor involved in regulating flowering, plant height and partial photomorphogenesis in dark.

KEY MESSAGE: OsiCRY2 is involved in light-regulated plant development and plays a role in regulating photomorphogenesis, plant height, flowering and most strikingly partial photomorphogenesis in dark. Cryptochrome 2 (CRY2), the blue/UV-A light photoreceptor in plants, has been reported to regulate photoperiod-dependent flowering and seedling photomorphogenesis (under low-intensity light). Among monocots, CRY2 has been reported from japonica rice, wheat, sorghum and barley. The two sub-species of rice, indica and japonica, exhibit a high degree of genetic variation and morphological and physiological differences. This article describes the characterization of CRY2 of indica rice (OsiCRY2). While the transcript levels of OsiCRY2 did not change significantly under blue light, its protein levels were found to decline with increased time duration under blue light. For phenotypic characterization, OsiCRY2 over-expression (OX) transgenics were generated in Oryza sativa Pusa Sugandh 2 (PS2) cultivar, a highly scented Basmati cultivar. The OsiCRY2OX transgenics displayed shorter coleoptiles and dwarfism than wild-type under blue light, white, and far-red light. Interestingly, even the dark-grown transgenics were shorter, concomitant with higher OsiCRY2 protein levels in transgenics than wild-type. Histological analysis revealed that the decrease in the length of the seedlings was due to a decrease in the length of the epidermal cells. The fully mature rice transgenics were shorter than the untransformed plants but flowered at the same time as wild-type. However, the OsiCRY2 Arabidopsis over-expressors exhibited early flowering by 10-15 days, indicating the potential and conservation of function of OsiCRY2. The whole-genome transcriptome profiling of rice transgenics revealed the differential up-regulation of several light-regulated genes in dark-grown coleoptiles. These data provide evidence that OsiCRY2 regulates photomorphogenesis, plant height, and flowering in indica rice.

Characterization of Cry2 genes (CRY2a and CRY2b) of B. napus and comparative analysis of BnCRY1 and BnCRY2a in regulating seedling photomorphogenesis.

Cryptochrome 2 (CRY2) perceives blue/UV-A light and regulates photomorphogenesis in plants. However, besides Arabidopsis, CRY2 has been functionally characterized only in native species of japonica rice and tomato. In the present study, the BnCRY2a, generating a relatively longer cDNA and harboring an intron in its 5'UTR, has been characterized in detail. Western blot analysis revealed that BnCRY2a is light labile and degraded rapidly by 26S proteasome when seedlings are irradiated with blue light. For functional analysis, BnCRY2a was over-expressed in Brassica juncea, a related species more amenable to transformation. The BnCRY2a over-expression (BnCRY2aOE) transgenics developed short hypocotyl and expanded cotyledons, accumulated more anthocyanin in light-grown seedlings, and displayed early flowering on maturity. Early flowering in BnCRY2aOE transgenics was coupled with the up-regulation of many flowering-related genes such as FT. The present study also highlights the differential light sensitivity of cry1 and cry2 in controlling hypocotyl elongation growth in Brassica. BnCRY2aOE seedlings developed much shorter hypocotyl under the low-intensity of blue light, while BnCRY1OE seedling hypocotyls were shorter under the high-intensity blue light, compared to untransformed seedlings.

Metabolomic Insights Into Endophyte-Derived Bioactive Compounds.

Among the various plant-associated microbiota, endophytes (the microbial communities inhabiting plant endosphere without causing disease symptoms) exhibit the most intimate and specific association with host plants. Endophytic microbes influence various aspects of plant responses (such as increasing availability of nutrients, tolerance against biotic and abiotic stresses, etc.) by modulating the primary and secondary metabolism of the host. Besides, endophytic microbes produce a diverse array of bioactive compounds, which have potential applications in the pharmaceutical, food, and cosmetic industries. Further, there is sufficient evidence for endophyte-derived plant metabolites, which could be pursued as alternative sources of commercially important plant metabolites. The field of bioprospecting, the discovery of novel chemistries, and endophyte-mediated production of plant metabolites have witnessed a boom with the advent of omics technologies (especially metabolomics) in endophyte research. The high throughput study of small metabolites at a particular timepoint or tissue forms the core of metabolomics. Being downstream to transcriptome and proteome, the metabolome provides the most direct reflection of the phenotype of an organism. The contribution of plant and microbial metabolomics for answering fundamental questions of plant-endophyte interaction, such as the effect of endophyte inoculation on plant metabolome, composition of metabolites on the impact of environmental stressors (biotic and abiotic), etc., have also been discussed.

Surface sterilization for isolation of endophytes: Ensuring what (not) to grow.

Endophytic microbiota opens a magnificent arena of metabolites that served as a potential source of medicines for treating a variety of ailments and having prospective uses in agriculture, food, cosmetics, and many more. There are umpteen reports of endophytes improving the growth and tolerance of plants. In addition, endophytes from lifesaving drug-producing plants such as Taxus, Nothapodytes, Catharanthus, and so forth have the ability to produce host mimicking compounds. To harness these benefits, it is imperative to isolate the true endophytes, not the surface microflora. The foremost step in endophyte isolation is the removal of epiphytic microbes from plant tissues, called as surface sterilization. The success of surface sterilization decides "what to grow" (the endophytes) and "what not to grow" (the epiphytes). It is very crucial to use an appropriate sterilant solution, concentration, and exposure time to ensure thorough surface disinfection with minimal damage to the endophytic diversity. Commonly used surface sterilants include sodium hypochlorite (2%-10%), ethanol (70%-90%), mercuric chloride (0.1%), formaldehyde (40%), and so forth. In addition, the efficiency could further be improved by pretreatment with surfactants such as Triton X-100, Tween 80, and Tween 20. This review comprehensively deals with the various sterilants and sterilization methods for the isolation of endophytic microbes. In addition, the mechanisms and rationale behind using specific surface sterilants have also been elaborated at length.

Electrical and Structural Properties of Semi-Polar-ZnO/a-Al2O3 and Polar-ZnO/c-Al2O3 Films: A Comparative Study.

In this work, the properties of ZnO films of 100 nm thickness, grown using atomic layer deposition (ALD) on a-(100) and c-(001) oriented Al2O3 substrate are reported. The films were grown in the same growth conditions and parameters at six different growth temperatures (Tg) ranging from 100 °C to 300 °C. All as-grown and annealed films were found to be polycrystalline, highly (001) oriented for the c-Al2O3 and highly (101) oriented for the a-Al2O3 substrate. The manifestation of semi-polar-(101) and polar (001)-oriented ZnO films on the same substrate provided the opportunity for a comparative study in terms of the influence of polarization on the electrical and structural properties of ZnO films. It was found that the concentration of hydrogen, carbon, and nitrogen impurities in polar (001)-oriented films was considerably higher than in semi-polar (101)-oriented ZnO films. The study showed that when transparent conductive oxide applications were considered, the ZnO layers could be deposited at a temperature of about 160 °C, because, at this growth temperature, the high electrical conductivity was accompanied by surface smoothness in the nanometer scale. On the contrary, semi-polar (101)-oriented films might offer a perspective for obtaining p-type ZnO films, because the concentration of carbon and hydrogen impurities is considerably lower than in polar films.

Exploiting endophytic microbes as micro-factories for plant secondary metabolite production.

Plant secondary metabolites have significant potential applications in a wide range of pharmaceutical, food, and cosmetic industries by providing new chemistries and compounds. However, direct isolation of such compounds from plants has resulted in over-harvesting and loss of biodiversity, currently threatening several medicinal plant species to extinction. With the breakthrough report of taxol production by an endophytic fungus of Taxus brevifolia, a new era in natural product research was established. Since then, the ability of endophytic microbes to produce metabolites similar to those produced by their host plants has been discovered. The plant "endosphere" represents a rich and unique biological niche inhabited by organisms capable of producing a range of desired compounds. In addition, plants growing in diverse habitats and adverse environmental conditions represent a valuable reservoir for obtaining rare microbes with potential applications. Despite being an attractive and sustainable approach for obtaining economically important metabolites, the industrial exploitation of microbial endophytes for the production and isolation of plant secondary metabolites remains in its infancy. The present review provides an updated overview of the prospects, challenges, and possible solutions for using microbial endophytes as micro-factories for obtaining commercially important plant metabolites.Key points• Some "plant" metabolites are rather synthesized by the associated endophytes.• Challenges: Attenuation, silencing of BGCs, unculturability, complex cross-talk.• Solutions: Simulation of in planta habitat, advanced characterization methods.

Structural Properties of Thin ZnO Films Deposited by ALD under O-Rich and Zn-Rich Growth Conditions and Their Relationship with Electrical Parameters.

The structural, optical, and electrical properties of ZnO are intimately intertwined. In the present work, the structural and transport properties of 100 nm thick polycrystalline ZnO films obtained by atomic layer deposition (ALD) at a growth temperature (Tg) of 100-300 °C were investigated. The electrical properties of the films showed a dependence on the substrate (a-Al2O3 or Si (100)) and a high sensitivity to Tg, related to the deviation of the film stoichiometry as demonstrated by the RT-Hall effect. The average crystallite size increased from 20-30 nm for as grown samples to 80-100 nm after rapid thermal annealing, which affects carrier scattering. The ZnO layers deposited on silicon showed lower strain and dislocation density than on sapphire at the same Tg. The calculated half crystallite size (D/2) was higher than the Debye length (LD) for all as grown and annealed ZnO films, except for annealed ZnO/Si films grown within the ALD window (100-200 °C), indicating different homogeneity of charge carrier distribution for annealed ZnO/Si and ZnO/a-Al2O3 layers. For as grown films the hydrogen impurity concentration detected via secondary ion mass spectrometry (SIMS) was 1021 cm-3 and was decreased by two orders of magnitude after annealing, accompanied by a decrease in Urbach energy in the ZnO/a-Al2O3 layers.

Targeted 16S rRNA gene and ITS2 amplicon sequencing of leaf and spike tissues of Piper longum identifies new candidates for bioprospecting of bioactive compounds.

Piper longum (also known as Indian long pepper) is widely used in Ayurvedic, Siddha and Unani medicine systems. The principle bioactive compound of this plant is piperine, which mainly accumulates in the fruits called spikes. The report of piperine production by endophytic microbes isolated from Piper sp., motivated us to investigate the endophytic microbial diversity associated with the spikes vis-à-vis leaves (which contain negligible levels of piperine). This is the first report to use metagenomics approach to unravel the endophytic microbial diversity in P. longum. Our results indicate that 2, 56, 631 bacterial OTUs and 1090 fungal OTUs were picked cumulatively from both the tissues. Although bacterial and fungal endophytes occupy the same niche, remarkable differences exist in their diversity and abundance. For instance, the most abundant bacterial genera in spikes were Nocardioides and Pseudonocardia (Phylum Actinobacteria; reported to produce bioactive compounds); while, in leaves were Larkinella and Hymenobacter (Phylum Bacteriodetes). Likewise, the fungal endophytes, Periconia, Cladosporium and Coniothyrium (which have been earlier reported to produce commercially important metabolites including piperine), were also present in high abundance in spikes, in comparison to leaves. Further, the results of PICRUSt analysis reveal the high metabolic potential of spike-associated bacteria for secondary metabolism, namely biosynthesis of alkaloids (including pyridine/piperidine), terpenes, flavonoids and antibiotics. Therefore, our findings indicate that the endophytes abundant or unique in spikes could be explored for bioprospecting of novel/commercially important metabolites; an approach that has both ecological and economical benefits.

Molecular and Functional Characterization of GR2-R1 Event Based Backcross Derived Lines of Golden Rice in the Genetic Background of a Mega Rice Variety Swarna.

Homozygous Golden Rice lines developed in the background of Swarna through marker assisted backcross breeding (MABB) using transgenic GR2-R1 event as a donor for the provitamin A trait have high levels of provitamin A (up to 20 ppm) but are dwarf with pale green leaves and drastically reduced panicle size, grain number and yield as compared to the recurrent parent, Swarna. In this study, we carried out detailed morphological, biochemical and molecular characterization of these lines in a quest to identify the probable reasons for their abnormal phenotype. Nucleotide blast analysis with the primer sequences used to amplify the transgene revealed that the integration of transgene disrupted the native OsAux1 gene, which codes for an auxin transmembrane transporter protein. Real time expression analysis of the transgenes (ZmPsy and CrtI) driven by endosperm-specific promoter revealed the leaky expression of the transgene in the vegetative tissues. We propose that the disruption of OsAux1 disturbed the fine balance of plant growth regulators viz., auxins, gibberellic acid and abscisic acid, leading to the abnormalities in the growth and development of the lines homozygous for the transgene. The study demonstrates the conserved roles of OsAux1 gene in rice and Arabidopsis.

Two New Trematodes of Family Acanthocolpidae Luhe, 1906 From Marine Fish Leiognathus daura (Cuvier) from the Coast of Puri, Orissa, India.

BACKGROUND: Genus Acanthocolpus (Trematoda: Acanthocolpiidae) is one of the most important zoonotic digenean with wide geographic distribution in the world. The purpose of the present study was to describe morphological and morphometrical characteristics of Acanthocolpus species, currently prevalent in marine fish fauna of Puri coast, Orissa, India. METHODS: Gastro-intestinal organs of Leiognathus daura (Cuvier) in Puri coast, Orissa, India, were examined for infectivity with digenean trematode species. For examination and measurements of helminthes, acetoalum carmine staining was performed, followed by camera Lucida drawings of morphological characters and measurements of morphometrical criteria with a calibrated microscope. Using valid trematode systematic keys, almost all the parasites were identified at the level of species. RESULTS: Overall, 36 marine fishes were found infected with at least one species of Acanthocolpus. Considering morphological characteristics of Acanthocolpus, two species were identified as new species including Acanthocolpus durghai sp.nov. and Acanthocolpus amrawatai sp.nov. CONCLUSION: During the survey of helminth parasites, collected six different species of the genus Acanthocolpus, out of these two are new species, another is redescribed to show certain variation, the new parasite was obtain from the intestine of marine fish Leiognathus daura (Cuvier).