Changes in Bacterial Diversity and Composition in Response to Co-inoculation of Arbuscular Mycorrhizae and Zinc-Solubilizing Bacteria in Turmeric Rhizosphere.

In the present study, the impact of co-inoculation of arbuscular mycorrhizal fungi (AM Rhizophagus sp., NCBI-MN710507) and Zinc solubilizing bacteria (ZSB2- Bacillus megaterium, NCBI-KY687496) on plant growth, soil dehydrogenase activity, soil respiration and the changes in bacterial diversity in rhizosphere of turmeric (Curcuma longa) were examined. Our results showed that higher plant height and dry biomass were observed in treatments co-inoculated with AM and ZSB2. Likewise, dehydrogenase activity and soil respiration were more significant in the co-inoculation treatment, indicating abundance of introduced as well as inherent microflora. Bacterial community analysis using 16S rRNA revealed changes in the structure and diversity of various taxa due to co-inoculation of AM and ZSB2. Alpha diversity indexes (Shannon and Chao1) and beta diversity indexes obtained through unweighted unifrac approach also showed variation among the treated samples. Chloroflexi was the dominant phylum followed by Proteobacteria, Actinobacteria and Acidobacteria which accounted for 80% of all treated samples. The composition of bacterial communities at genus level revealed that co-inoculation caused distinct bacterial profiles. The Linear discriminant analysis effect size revealed the dominance of ecologically significant genera such as Bradyrhizobium, Candidatus, Pedomicrbium, Thermoporothrix, Acinetobacter and Nitrospira in treatments co-inoculated with AM and ZSB2. On the whole, co-inoculated treatments revealed enhanced microbial activities and caused significant positive shifts in the bacterial diversity and abundance compared to treatments with sole application of ZSB2 or AM.

A Single Amino Acid Substitution in an ORANGE Protein Promotes Carotenoid Overaccumulation in Arabidopsis.

Carotenoids are crucial for plant growth and human health. The finding of ORANGE (OR) protein as a pivotal regulator of carotenogenesis offers a unique opportunity to comprehensively understand the regulatory mechanisms of carotenoid accumulation and develop crops with enhanced nutritional quality. Here, we demonstrated that alteration of a single amino acid in a wild-type OR greatly enhanced its ability to promote carotenoid accumulation. Whereas overexpression of OR from Arabidopsis (Arabidopsis thaliana; AtOR) or from the agronomically important crop sorghum (Sorghum bicolor; SbOR) increased carotenoid levels up to 2-fold, expression of AtOR(His) (R90H) or SbOR(His) (R104H) variants dramatically enhanced carotenoid accumulation by up to 7-fold in the Arabidopsis calli. Moreover, we found that AtOR(Ala) (R90A) functioned similarly to AtOR(His) to promote carotenoid overproduction. Neither AtOR nor AtOR(His) greatly affected carotenogenic gene expression. AtOR(His) exhibited similar interactions with phytoene synthase (PSY) as AtOR in posttranscriptionally regulating PSY protein abundance. AtOR(His) triggered biogenesis of membranous chromoplasts in the Arabidopsis calli, which shared structures similar to chromoplasts found in the curd of the orange cauliflower (Brassica oleracea) mutant. By contrast, AtOR did not cause plastid-type changes in comparison with the controls, but produced plastids containing larger and electron-dense plastoglobuli. The unique ability of AtOR(His) in mediating chromoplast biogenesis is responsible for its induced carotenoid overproduction. Our study demonstrates OR(His/Ala) as powerful tools for carotenoid enrichment in plants, and provides insights into the mechanisms underlying OR(His)-regulated carotenoid accumulation.

Isolation and characterization of genomic microsatellite markers for small cardamom (Elettaria cardamomum Maton) for utility in genetic diversity analysis.

Microsatellite markers in small cardamom (Elettaria cardamomum Maton) were developed using the selective hybridization enrichment method. A total of 140 microsatellite repeats were identified from 270 clones. Primers were designed for 58 microsatellites and 44 primer pairs amplified products of expected size in cardamom. These markers were used for studying the diversity of 20 important small cardamom genotypes, and six markers were found to be polymorphic. The number of alleles ranged from 2 to 7 with an average of 3.6 per locus. Polymorphic information content values ranged from 0.14 to 0.38 based on dominant scoring. The two markers ECM 47a and ECMG 28 generated specific banding patterns for the genotypes MCC7 (Pink tiller) and APG434 (MA18) respectively. Dendrogram illustrated the genetic similarity between different genotypes of Kerala and Karnataka regions. It differentiated the closely related genotypes and released varieties into separate groups. Principal coordinate analysis revealed PV1 and ICRI 1 as the most divergent genotypes. The study demonstrated that these markers are informative and can be further utilized for generating reliable molecular data for assisting the crop improvement of small cardamom. Cross generic transferability (71.4 %) of the developed primers proved that they are useful for phylogenetic studies in the family Zingiberaceae. This is the first report of de novo isolation, characterisation and utilization of microsatellite markers for the genetic diversity analysis of small cardamom.

Detection of plant-based adulterants in turmeric powder using DNA barcoding.

CONTEXT: In its powdered form, turmeric [Curcuma longa L. (Zingiberaceae)], a spice of medical importance, is often adulterated lowering its quality. OBJECTIVE: The study sought to detect plant-based adulterants in traded turmeric powder using DNA barcoding. MATERIALS AND METHODS: Accessions of Curcuma longa L., Curcuma zedoaria Rosc. (Zingiberaceae), and cassava starch served as reference samples. Three barcoding loci, namely ITS, rbcL, and matK, were used for PCR amplification of the reference samples and commercial samples representing 10 different companies. PCR success rate, sequencing efficiency, occurrence of SNPs, and BLAST analysis were used to assess the potential of the barcoding loci in authenticating the traded samples of turmeric. RESULTS: The PCR and sequencing success of the loci rbcL and ITS were found to be 100%, whereas matK showed no amplification. ITS proved to be the ideal locus because it showed greater variability than rbcL in discriminating the Curcuma species. The presence of C. zedoaria could be detected in one of the samples whereas cassava starch, wheat, barley, and rye in other two samples although the label claimed nothing other than turmeric powder in the samples. DISCUSSION AND CONCLUSION: Unlabeled materials in turmeric powder are considered as adulterants or fillers, added to increase the bulk weight and starch content of the commodity for economic gains. These adulterants pose potential health hazards to consumers who are allergic to these plants, lowering the product's medicinal value and belying the claim that the product is gluten free. The study proved DNA barcoding as an efficient tool for testing the integrity and the authenticity of commercial products of turmeric.

A simple and efficient protocol for isolation of high quality functional RNA from different tissues of turmeric (Curcuma longa L.).

Many experiments in plant molecular biology require processing of a large number of RNA samples and in some cases large quantities are required for a single application. In turmeric, a major spice and medicinal plant, a protocol for RNA isolation is not available. The major difficulty encountered while using other popular protocols is the low yield and quality of RNA which hampers the downstream applications like qRT-PCR, cDNA synthesis and micro RNA isolation. Commercial kits though available are costly and were found to be unsuccessful in case of rhizomes and root tissues that are rich in polyphenols, polysaccharides and alkaloids. It was thus felt that a quick, handy and cheap protocol of total RNA isolation from different tissues of turmeric was required for day to day working in our lab. The new protocol utilizes SDS based extraction buffer including ß-mercaptoethanol and PVP with sequential acid phenol:chloroform extraction to remove polyphenols and proteins, followed by the purification with sodium acetate to eliminate polysaccharides. The protocol is simple and can be completed in less than 3 h. The RNA yield from rhizome was higher by more than fivefold with both A260/280 and A260/230 ratio in the range of 1.8-2.0. The protocol worked well with leaf, rhizome, pseudostem and root tissues with RIN >7.0 and the isolated RNA could be successfully used for cDNA synthesis, RT-PCR, qRT-PCR and small RNA isolation including microRNA.

Whole genome based identification of BAHD acyltransferase gene involved in piperine biosynthetic pathway in black pepper.

Black pepper (Piper nigrum L.), a crop of the genus Piper, is an important spice that has both economic and ecological significance. It is widely regarded as the "King of Spices" because of its pungency, attributed to the presence of piperine. BAHD acyl transferase, the crucial enzyme involved in the final step in piperine biosynthesis was the focus of our study and the aim was to identify the candidate isoform involved in biosynthesis of piperine. Reference genome-based analysis of black pepper identified six BAHD-AT isoforms and mapping of these sequences revealed that the isoforms were situated on six distinct chromosomes. By using specific primers for each of these transcripts, qPCR analysis was done in different tissues as well as berry stages to obtain detectable amplification products. Expression profiles of isoforms from chromosome 6 correlated well with piperine content compared to other five isoforms, across tissues and was therefore assumed to be involved in biosynthesis of piperine. In addition to this, we could also identify the binding sites of MYB transcription factor in the cis-regulatory regions of the isoforms. We also used in-silico docking and molecular dynamics simulation to calculate the binding free energy of the ligand and confirmed that among all the isoforms, BAHD-AT from chromosome 6 had the lowest free binding energy and highest affinity towards the ligand. Our findings are expected to aid the identification of new genes connecting enzymes involved in the biosynthetic pathway of piperine, which will have major implications for future research in metabolic engineering.Communicated by Ramaswamy H. Sarma.

RAPD, SCAR and conserved 18S rDNA markers for a red-listed and endemic medicinal plant species, Knema andamanica (Myristicaceae).

Knema andamanica is a red-listed endemic medicinal species of Myristicaceae restricted to Andaman and Nicobar (A&N) Islands, India. This species is used in tribal medicines and has immense bioprospective potential. With a view to generate suitable genomic markers for classification and identification, we have generated RAPD, SCAR and conserved 18S rDNA markers from K. andamanica. A unique 585 bp fragment, that distinguished it from seven other related species of Myristicaceae was first amplified using the random primer OPE 06 and converted to SCAR marker (GenBank accession # JN228256). The conserved sequences of 18S rDNA loci from K. andamanica were also amplified and sequenced (GenBank accession #JN228265). The sequence revealed deviations including 18 variable regions and 15 indels that were unique to K. andamanica. These markers can help in definite identification of K. andamanica even at the juvenile stages.

Metagenomics indicates abundance of biofilm related genes and horizontal transfer of multidrug resistant genes among bacterial communities in nano zinc oxide polluted soil.

The unsafe and reckless disposal of metal oxide nanoparticles like ZnO (nZnO) into the soil could seriously impact bacterial behavioural responses and functions. Under such stress, biofilm formation is considered to be a robust mechanism for bacterial survival in soil. We examined the response of bacterial metagenomes in soils exposed to varying levels of Zn (50, 200, 500 and 1000 mg kg-1) as nano Zn oxide (nZnO) in terms of biofilm genesis and regulation and their co-occurrences with multidrug resistance genes (MDRGs) and mobile genetic elements (MGEs). The size-specific effects of nZnO were verified using its bulk counterpart (bZnO). Both nZnO and bZnO facilitated profusion of biofilm related genes (BGs) especially at higher Zn levels (500 and 1000 mg kg-1 Zn), though maximum abundance was registered at a comparatively lower level under nZnO. In general, nZnO favoured an enhancement of genes involved in exopolysaccharide biosynthesis and attachment, while bZnO favoured genes related to capsule formation, chemotaxis and biofilm dispersion. Co-occurrence network analysis revealed significant positive correlations between abundances of BGs, MDRGs and MGEs, indicating an enhanced probability for horizontal gene transfer of MDRGs in nZnO polluted soils.

Soil polluted with nano ZnO reveals unstable bacterial communities and decoupling of taxonomic and functional diversities.

Due to relentless production and disposal of nano zinc oxide (nZnO), it has become critical to comprehend the serious risks large-scale accumulation of nZnO pose to bacterial communities in soil. The primary objective was to evaluate the changes in bacterial community structure and associated functional pathways through predictive metagenomic profiling and subsequent validation through Quantitative Realtime PCR in soil spiked with nZnO (0, 50, 200, 500 and 1000 mg Zn kg-1) and similar levels of bulk ZnO (bZnO). The results revealed that soil microbial biomass-C, -N, -P, soil respiration and enzyme activities decreased markedly at higher ZnO levels. The alpha diversity decreased with increasing ZnO level, with more impact under nZnO, while beta diversity analyses indicated a distinct dose- dependent separation of bacterial communities. The dominant taxa including Proteobacteria, Bacterioidetes, Acidobacteria and Planctomycetes significantly increased in abundance, while Firmicutes, Actinobacteria and Chloroflexi decreased in abundance with elevated nZnO and bZnO levels. Redundancy analysis indicated that changes in bacterial community structure instilled a greater dose- rather than size- specific response on key microbial parameters. Predicted key functions did not show a dose- specific response, and at 1000 mg Zn kg-1, methane metabolism as well as starch and sucrose metabolism were attenuated, while functions involving two component systems and bacterial secretion systems were enhanced under bZnO indicating better stress avoidance mechanism than under nZnO. Realtime PCR and microbial endpoint assays confirmed the metagenome derived taxonomic and functional data, respectively. Taxa and functions that varied substantially under stress were established as bioindicators to predict nZnO toxicity in soils. Taxon-function decoupling indicated that the soil bacterial communities deployed adaptive mechanisms under high ZnO, with lesser buffering capacity and resilience of communities under nZnO.

Dataset of De Novo hybrid berry transcriptome profiling and characterization of Piper species (Piper nigrum and Piper longum) using Illumina and Nanopore sequencing.

Piper nigrum and Piper longum are the most popular and economically essential spice crops globally valued for their aromatic alkaloids, especially Piperine. However, Piperine synthesis pathway mechanisms are not yet well known. This work was aimed to generate the full-length comparative berry transcriptome analysis dataset of P. nigrum and P. longum by Illumina and Nanopore sequencing platforms. While short-read sequencing technology is widely using to capture transcriptome profiles, there are still some limitations due to the read length. We used Oxford Nanopore technology for long reads and the Illumina sequencing platform for short reads to generate a hybrid transcriptome assembly from half matured and fully matured berries of P. nigrum and P. longum. From P. nigrum and P. longum 37.3 million and 38.1 million raw reads were generated respectively. A total of 308369 contigs from P. nigrum and 267715 contigs from P. longum were obtained and successfully annotated. The transcriptome data revealed gene families involved in piperine and other secondary metabolite biosynthetic pathways. The raw data were uploaded to NCBI database. This dataset shed light on the further exploration of the piperine biosynthetic pathway, its transcriptomic changes, and evolution. Data generated has been submitted to SRA of NCBI with Bio samples accession: (SAMN13981803, SAMN22826456).

Development and characterization of salt tolerant somaclones in rice cultivar pokkali.

Primary regenerants (1190) of a tall traditional salt tolerant rice cultivar pokkali were produced through in vitro culture from mature seed derived calli of fourth subculture. Out of 35000 SC2 regenerants, 26 promising lines with superior agronomic traits were chosen initially for evaluation. SC3 and SC4 generations were stringently evaluated under hydroponics with excess salt stress as well as under field conditions across two growing seasons in Bay Islands. A set of 10 promising somaclones was further evaluated at SC5 and SC6 of which BTS 2, BTS 13, BTS 18 and BTS 24 were found promising. In SC7 and SC8 yield trials in research farm, BTS 24 was found to produce a mean yield of 36.3 and 45.9 q ha-1 under saline and normal soil conditions, respectively. Somaclones varied significantly from the parent with respect to yield and yield attributes. Grain quality and biochemical parameters of all elite somaclones were different from the parent. However, somaclones did not deviate much from their parent in respect of disease and insect pest resistance pattern.

Assessment of androclonal variation in an indica rice PTB 28.

Anther culture (AC) derived 160 doubled haploid (DH) families of an indica rice PTB 28 were field evaluated at Port Blair (lat 11 degrees.41'13.04"N; long 92 degrees 43'30.16"E), to assess the extent and range of induced variability among the androclones at segregating A2 generation and identify promising selectants for genetic improvement. Though the coefficient of variation (CV) for plant height was least (2.65%), majority of the androclones were shorter (81.7%). CV was high in case of panicle number per plant (24.59%) and tiller number per plant (28.28%), while it was maximum in respect of yield per plant (37.0%). Tiller and panicle number per plant registered maximum reduction compared to parent, however, displayed larger variations in double haploid (DH) lines. Out of 160 DH families, only 10 (6.3%) lines out-yielded the parent. Extreme types with wide variations were encountered which reaffirms the possibility of employing anther culture in rice improvement by restoring yield advantage in advanced generations or by tailoring them through recombination breeding.

Development, characterization and cross species amplification of polymorphic microsatellite markers from expressed sequence tags of turmeric (Curcuma longa L.).

Expressed sequence tags (ESTs) from turmeric (Curcuma longa L.) were used for the screening of type and frequency of Class I (hypervariable) simple sequence repeats (SSRs). A total of 231 microsatellite repeats were detected from 12,593 EST sequences of turmeric after redundancy elimination. The average density of Class I SSRs accounts to one SSR per 17.96 kb of EST. Mononucleotides were the most abundant class of microsatellite repeat in turmeric ESTs followed by trinucleotides. A robust set of 17 polymorphic EST-SSRs were developed and used for evaluating 20 turmeric accessions. The number of alleles detected ranged from 3 to 8 per loci. The developed markers were also evaluated in 13 related species of C. longa confirming high rate (100%) of cross species transferability. The polymorphic microsatellite markers generated from this study could be used for genetic diversity analysis and resolving the taxonomic confusion prevailing in the genus.