Modulation of terpenoid indole alkaloid pathway via elicitation with phytosynthesized silver nanoparticles for the enhancement of ajmalicine, a pharmaceutically important alkaloid.

MAIN CONCLUSION: The use of silver nanoparticles as elicitors in cell cultures of Rauwolfia serpentina resulted in increased levels of ajmalicine, upregulated structural and regulatory genes, elevated MDA content, and reduced activity of antioxidant enzymes. These findings hold potential for developing a cost-effective method for commercial ajmalicine production. Plants possess an intrinsic ability to detect various stress signals, prompting the activation of defense mechanisms through the reprogramming of metabolites to counter adverse conditions. The current study aims to propose an optimized bioprocess for enhancing the content of ajmalicine in Rauwolfia serpentina callus through elicitation with phytosynthesized silver nanoparticles. Initially, callus lines exhibiting elevated ajmalicine content were established. Following this, a protocol for the phytosynthesis of silver nanoparticles using seed extract from Rauwolfia serpentina was successfully standardized. The physicochemical attributes of the silver nanoparticles were identified, including their spherical shape, size ranging from 6.7 to 28.8 nm in diameter, and the presence of reducing-capping groups such as amino, carbonyl, and amide. Further, the findings indicated that the presence of 2.5 mg L-1 phytosynthesized silver nanoparticles in the culture medium increased the ajmalicine content. Concurrently, structural genes (TDC, SLS, STR, SGD, G10H) and regulatory gene (ORCA3) associated with the ajmalicine biosynthetic pathway were observed to be upregulated. A notable increase in MDA content and a decrease in the activities of antioxidant enzymes were observed. A notable increase in MDA content and a decrease in the activities of antioxidant enzymes were also observed. Our results strongly recommend the augmentation of ajmalicine content in the callus culture of R. serpentina through supplementation with silver nanoparticles, a potential avenue for developing a cost-effective process for the commercial production of ajmalicine.

A glossy mutant in onion (Allium cepa L.) shows decreased expression of wax biosynthesis genes.

Cuticular wax is a characteristic feature of land plants that provides protection against both biotic and abiotic stresses. In this study, a glossy mutant lacking an epicuticular wax layer was identified in the γ-irradiated M2 mutant population of the onion cultivar Bhima Super. The inheritance of the mutant's glossy phenotype was determined to be recessive and single locus. Scanning electron microscopy analysis showed poor accumulation of wax crystals in the glossy mutant, concentrated near the stomata. The plant height, number of leaves per plant, and stomatal parameters of the mutant were similar to the wild-type. RNA-seq was used to comprehend the expression variations of waxy cuticle-related genes in the glossy mutant and its wild-type waxy cultivars. Differential gene expression analysis of the RNA-seq data revealed that the genes involved in wax biosynthesis, such as AcCER1, AcCER26, AcMAH1, and AcWSD1, were downregulated by 2.72, 1.74, 2.59 and 2.12-fold, respectively, in the glossy mutant respectively. The expression patterns of these four unigenes were validated using semi-quantitative RT-PCR. The glossy mutant displayed a substantial 3.5-fold reduction in cuticular wax load compared to the wild-type due to the significant downregulation of these wax biosynthesis genes. These findings represent early advancements in understanding the molecular mechanisms of wax biosynthesis in onions. Furthermore, they provide a foundation for utilizing the glossy mutant trait in breeding programmes to enhance stress and pest resilience.

CRISPR/Cas9-mediated editing of PHYTOENE DESATURASE gene in onion (Allium cepa L.).

Introduction: Clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) is a precise genome editing tool used to introduce genetic modifications in a wide range of crop species. Thus far, there is no report of CRISPR/Cas9-mediated genome editing in onions (Allium cepa L.). Methods: In the present study, we targeted two exons of the gene coding for Phytoene desaturase (AcPDS) in onion cv. Bhima Super. The sgRNA-carrying constructs were co-cultivated with 8-week-old embryogenic calli using an Agrobacterium-mediated transformation protocol and incubated on the media without hygromycin B selection. Results and discussion: Out of the total 617 co-cultivated calli, 21 (3.4%) regenerated shoots exhibited three distinct phenotypes: albino, chimeric, and pale green; in comparison to the wild-type non-transformed regenerated shoots. Total chlorophyll content was drastically reduced in albino shoots and significantly decreased in chimeric shoots. Out of the six Cas9 gene PCR-confirmed regenerated shoots, two exhibited the albino phenotype due to insertions/deletions (InDels) and substitution-based mutations in and around the AcPDS target sites. Deep amplicon sequencing revealed a significantly variable InDel frequency between two sgRNAs, ranging from 1.2% to 63.4%, along with a 53.4% substitution frequency. The mutation of the AcPDS gene generated a visually detectable albino phenotype, thus confirming the successful editing of the AcPDS gene. This is the first time a CRISPR/Cas9-mediated genome editing protocol has been successfully established in onion, with the AcPDS gene serving as an example. This study will provide the necessary momentum for researchers to further basic and applied research on onions.

Identification, cloning and characterization of AcMSH1 from Onion (Allium cepa L.).

BACKGROUND: MSH1 (MutS homolog1) is a nuclear-encoded protein that plays a crucial role in maintaining low mutation rates and stability of the organellar genome. While plastid MSH1 maintains nuclear epigenome plasticity and affects plant development patterns, mitochondrial MSH1 suppresses illegitimate recombination within the mitochondrial genome, affects mitochondrial genome substoichiometric shifting activity and induces cytoplasmic male sterility (CMS) in crops. However, a detailed functional investigation of onion MSH1 has yet to be achieved. MATERIALS AND RESULTS: The homology analysis of onion genome database identified a single copy of the AcMSH1 gene in the onion cv. Bhima Super. In silico analysis of AcMSH1 protein sequence revealed the presence of 6 conserved functional domains including a unique MSH1-specific GIY-YIG endonuclease domain at the C-terminal end. At N-terminal end, it has signal peptide sequences targeting chloroplast and mitochondria. The concentration of AcMSH1 was found to be highest in isolated mitochondria, followed by chloroplasts, and negligible in the cytoplasmic fraction; which proved its localization to the mitochondria and chloroplasts. Quantitative expression analysis revealed that AcMSH1 protein levels were highest in leaves, followed by flower buds, root tips, flowers, and umbels, with the lowest amount found in callus tissue. CONCLUSION: Onion genome has single copy of MSH1, with characteristic GIY-YIG endonuclease domain. AcMSH1 targeted towards both chloroplasts and mitochondria. The identification and characterisation of AcMSH1 may provide valuable insights into the development of CMS lines in onion.

A simple flow cytometry-based assay to study global methylation levels in onion, a non-model species.

DNA methylation is an important epigenetic mark and global methylation dynamics regulate plant developmental processes. Even though genome sequencing technologies have made DNA methylation studies easier, it is difficult in non-model species where genome information is not available. Therefore in this study, we developed a simple assay for analysing global methylation levels in plants by washless immunolabelling of unfixed nuclei using flow cytometry. Onion leaf tissue was used as a model system, and mean fluorescence intensity due to anti-5- methyl cytosine (5-mC) antibodies were used as a measure of global methylation levels. Among three nuclear isolation buffers evaluated, the highest nuclear yield with the low background was obtained with LB01. To maintain a balance between high DNA fluorescence value and low coefficient of variation of DNA peaks, 45 min of hydrolysis with 0.2 N hydrochloric acid was used for chromatin denaturation resulting in six-fold increase in 5-mC fluorescence compared to control. This method was used successfully to detect 5-Azacytidine induced DNA hypomethylation in onion leaf tissues. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01047-6.

Microbiome profiling of the onion thrips, Thrips tabaci Lindeman (Thysanoptera: Thripidae).

The gut microbial community structure of adult Thrips tabaci collected from 10 different agro-climatically diverse locations of India was characterized by using the Illumina MiSeq platform to amplify the V3 region of the 16S rRNA gene of bacteria present in the sampled insects. Analyses were performed to study the bacterial communities associated with Thrips tabaci in India. The complete bacterial metagenome of T. tabaci was comprised of 1662 OTUs of which 62.25% belong to known and 37.7% of unidentified/unknown bacteria. These OTUs constituted 21 bacterial phyla of 276 identified genera. Phylum Proteobacteria was predominant, followed by Actinobacteria, Firmicutes, Bacteroidetes and Cyanobacteria. Additionally, the occurrence of the reproductive endosymbiont, Wolbachia was detected at two locations (0.56%) of the total known OTUs. There is high variation in diversity and species richness among the different locations. Alpha-diversity metrics indicated the higher gut bacterial diversity at Bangalore and lowest at Rahuri whereas higher bacterial species richness at T. tabaci samples from Imphal and lowest at Jhalawar. Beta diversity analyses comparing bacterial communities between the samples showed distinct differences in bacterial community composition of T. tabaci samples from different locations. This paper also constitutes the first record of detailed bacterial communities associated with T. tabaci. The location-wise variation in microbial metagenome profile of T. tabaci suggests that bacterial diversity might be governed by its population genetic structure, environment and habitat.

Ectopic Expression of Cold Responsive LlaCIPK Gene Enhances Cold Stress Tolerance in Nicotiana tabacum.

Low-temperature stress severely affects the growth, development, and geographical distribution of various crop plants, resulting in significant economic loss to producers. In a quest to identify cold-regulated genes, we constructed a cDNA suppression subtractive library from a high altitude adapted ecotype of Lepidium. We cloned a cold-induced gene LlaCIPK from the subtracted cDNA library which gave homology to Arabidopsis CIPK15 gene. The predicted 3D structure of LlaCIPK protein also showed homology with Arabidopsis CIPK protein. Quantitative real-time PCR analysis in Lepidium seedlings exposed to 6 h of cold stress shows a 3-fold increase in the expression of LlaCIPK transcript. The expression of LlaCIPK was also differentially regulated by ethylene, CaCl2, ABA, and SA treatments. Ethylene and CaCl2 treatments up regulated LlaCIPK expression, whereas ABA and SA treatments down regulated the LlaCIPK expression. Transgenic plants overexpressing LlaCIPK gene under constitutive promoter show an increased level of proline and cell membrane stability. Taken together, our results suggest that the LlaCIPK contributes to the cold-response pathway in Lepidium plants.

Construction of cold induced subtracted cDNA library from Cicer microphyllum and transcript characterization of identified novel wound induced gene.

A forward cold induced subtracted cDNA library was constructed to identify the stress regulated genes in a high altitude cold desert-adapted species Cicer microphyllum, a wild relative of cultivated chickpea, distributed in western and trans-Himalayas. A total of 1,040 clones were obtained from the subtracted cDNA library. These clones were screened for presence of insert with colony PCR. A total of 523 clones were picked by colony PCR among which 300 clones were observed differentially expressed as per dot blot analysis. Differentially expressed clones were sequenced and assembled into clusters based on the presence of overlapping, identical, or similar sequences. A total of 283 ESTs were submitted in gene bank (accession numbers GO241043 to GO241326). BLAST analysis of these ESTs revealed its similarity for regulatory proteins like kinases, metallothionin, and enzymes/proteins with unknown functions. A cDNA encoding wound induced like protein, identified from this cold induced subtraction cDNA library, was full-length cloned using RACE and sequenced (accession number GQ914056). Southern blot of C. microphyllum indicated single copy of the gene in genome. Transcript expression profiling of this gene by quantitative real-time PCR and northern blot confirmed its up-regulation during low temperature stress. Further, in situ RNA hybridization also revealed cold (4°C) induced expression of the gene.

Isolation and characterization of cold responsive NAC gene from Lepidium latifolium.

Cold stress is one of the major limiting factor in crop productivity. Plants growing in colder regions acclimatize to severe conditions owing to the presence of 'cold stress tolerant genes'. Isolation and functional characterization of these genes are important before their exploitation in modern agricultural practices. Here, we have cloned full length NAC gene (1,388 bp) from Lepidium latifolium (LlaNAC). This gene belongs to NAP sub-group which also includes ANAC056 of Arabidopsis thaliana, nearest relative of LlaNAC. Upstream analysis and microarray data analysis of ANAC056 suggested that LlaNAC might also be ABA-regulated. However, quantitative transcript expression analysis revealed that LlaNAC transcript upregulated by cold stress and downregulated in response to varying concentrations of abscisic acid, salicylic acid, calcium chloride and ethylene. There is also a possibility that the gene may be getting regulated by a pathway whose components are still unknown. Any further investigations to understand the mechanism of regulation of LlaNAC gene expression are likely to find immense importance in plant biotechnology and crop improvement.

Induced ectopic expression of At-CBF1 in marker-free transgenic tomatoes confers enhanced chilling tolerance.

In an attempt to improve chilling stress tolerance, an Arabidopsis C-repeat binding factor 1 (At-CBF1) gene driven by the inducible promoter RD29A was co-transferred into tomato var. Shalimar. Marker (NPTII)-free transgenic were obtained in T(1) generation because of unlinked integration of CBF1 and NPTII genes. Reverse transcription-polymerase chain reaction confirmed the expression of CBF1 in T(1) transgenic lines. Study of expression pattern in T(1) transgenic line showed a gradual increase with increasing chilling stress period and also confirmed the reversibility of expression on removal of stress. The transgenic plants exhibited no morphological and agronomical differences as compared to non-transformed plants. When young transgenic plants were exposed to chilling stress (4°C) for 3 days, increased survival (50%) was observed in transgenic lines than non-transformed plants (10%). Transgenic plants subjected to the chilling stress showed a significant decrease in membrane injury index and lipid peroxidation and also increased significantly free proline content in the leaf tissues as compared to non-transformed plants. Thus, these findings indicate that marker-free transgenic tomato plants expressing Arabidopsis CBF1 gene provided protection and conferred cold tolerance to transgenic tomato without any phenotypic variation.

Metallothionein-like gene from Cicer microphyllum is regulated by multiple abiotic stresses.

Cicer microphyllum, a wild relative of cultivated chickpea, is a high altitude cold desert-adapted species distributed in western and trans-Himalayas. A complementary DNA (cDNA) encoding metallothionein-like protein has been identified from a cold-induced subtraction cDNA library from C. microphyllum. The sequence of the cloned metallothionein gene from C. microphyllum (GQ900702) contains 240-bp-long open reading frame and encodes predicted 79-amino acid protein of 7.9 kDa. Sequence analysis identified the motifs characteristic of type II metallothionein and designated as CmMet-2. Southern hybridization confirms a single copy of the CmMet-2 gene in C. microphyllum genome. In situ hybridization indicated spatial transcript regulation of CmMet-2 in root and aerial parts and also confirmed through real-time PCR-based quantitative transcript analysis. The data revealed a significantly low level of transcript in the aerial parts than the roots. Quantitative analysis using real-time PCR assay revealed induction of transcript in all parts of plants in response to cold stress at 4°C. The transcript abundance was found to increase exponentially with time course from 6 to 24 h after exposure. Further, regulation of transcript accumulation in response to abscisic acid application, polyethylene glycol (100 µM)-induced osmotic stress, or ZnSO(4) (1 µM) foliar spray indicated by Northern hybridization suggests the involvement of CmMet-2 in multiple stress response.