Engineering medicinal plant-derived CYPs: a promising strategy for production of high-valued secondary metabolites.

MAIN CONCLUSION: Cytochorme P450s (CYPs) play a critical role in the catalysis of secondary metabolite biosynthetic pathways. For their commercial use, various strategies for metabolic pathway engineering using CYP as a potential target have been explored. Plants produce a vast diversity of secondary metabolites which are being used to treat various ailments and diseases. Some of these metabolites are difficult to obtain in large quantities limiting their industrial use. Cytochrome P450 enzymes (CYPs) are important catalysts in the biosynthesis of highly valued secondary metabolites, and are found in all domains of life. With the development of high-throughput sequencing and high-resolution mass spectrometry, new biosynthetic pathways and associated CYPs are being identified. In this review, we present CYPs identified from medicinal plants as a potential game changer in the metabolic engineering of secondary metabolic pathways. We present the achievements made so far in enhancing the production of important bioactivities through pathway engineering, giving some popular examples. At last, current challenges and possible strategies to overcome the limitations associated with CYP engineering to enhance the biosynthesis of target secondary metabolites are also highlighted.

Identification and characterization of chickpea genotypes for early flowering and higher seed germination through molecular markers.

BACKGROUND: Chickpea is the fourth most important legume crop contributing 15.42% to the total legume production and a rich source of proteins, minerals, and vitamins. Determination of genetic diversity of wild and elite cultivars coupled with early flowering and higher seed germination lines are quintessential for variety improvement. METHODS AND RESULTS: In the present study, we have analyzed the genetic diversity, population structure, cross-species transferability, and allelic richness in 50 chickpea collections using 23 Inter simple sequence repeats (ISSR) markers. The observed parameters such as allele number varied from 3 to 16, range of allele size varied from 150 to 1600 bp and polymorphic information content (PIC) range lies in between 0.15 and 0.49. Dendrogram was constructed with ISSR marker genotypic data and classified 50 chickpea germplasms into groups I and II, where the accession P 74 - 1 is in group I and the rest are in group II. Dendrogram, Principal component analysis (PCA), dissimilarity matrix, and Bayesian model-based genetic clustering of 50 chickpea germplasms revealed that P 74 - 1 and P 1883 are very diverse chickpea accessions. CONCLUSION: Based on genetic diversity analysis, 15 chickpea germplasm having been screened for early flowering and higher seed germination and found that the P 1857-1 and P 3971 have early flowering and higher seed germination percentage in comparison to P 1883 and other germplasm. These agronomic traits are essential for crop improvement and imply the potential of ISSR markers in crop improvement.

Genome-wide analysis of RING-type E3 ligase family identifies potential candidates regulating high amylose starch biosynthesis in wheat (Triticum aestivum L.).

In ubiquitin-mediated post-translational modifications, RING finger families are emerged as important E3 ligases in regulating biological processes. Amylose and amylopectin are two major constituents of starch in wheat seed endosperm. Studies have been found the beneficial effects of high amylose or resistant starch on health. The ubiquitin-mediated post-translational regulation of key enzymes for amylose/amylopectin biosynthesis (GBSSI and SBEII) is still unknown. In this study, the genome-wide analysis identified 1272 RING domains in 1255 proteins in wheat, which is not reported earlier. The identified RING domains classified into four groups-RING-H2, RING-HC, RING-v, RING-G, based on the amino acid residues (Cys, His) at metal ligand positions and the number of residues between them with the predominance of RING-H2 type. A total of 1238 RING protein genes were found to be distributed across all 21 wheat chromosomes. Among them, 1080 RING protein genes were identified to show whole genome/segmental duplication within the hexaploid wheat genome. In silico expression analysis using transcriptome data revealed 698 RING protein genes, having a possible role in seed development. Based on differential gene expression and correlation analysis of 36 RING protein genes in diverse (high and low) amylose mutants and parent, 10 potential RING protein genes found to be involved in high amylose biosynthesis and significantly associated with two starch biosynthesis genes; GBSSI and SBEIIa. Characterization of mutant lines using next-generation sequencing method identified unique mutations in 698 RING protein genes. This study signifies the putative role of RING-type E3 ligases in amylose biosynthesis and this information will be helpful for further functional validation and its role in other biological processes in wheat.

Genome-wide identification and characterization of novel non-coding RNA-derived SSRs in wheat.

Expression of eukaryotic genes is largely regulated by non-coding RNAs (ncRNA). Sequence variations in the regulatory RNAs may have critical biological consequences including transcriptional and post-transcriptional gene regulation. ncRNA-derived markers thus can be proved useful in molecular breeding, QTL mapping and association studies for trait dissection. In present study, we identified a total of 661 SSRs dwelling in pre-miRNA (15), small nuclear RNA (25) and lncRNA (621). Of these, 46 were validated and 100% amplification success was observed in selected wheat genotypes. A set of 36 ncRNA-SSRs markers was utilized for genetic variability assessment in forty-eight Indian wheat genotypes (which includes bread wheat, durum wheat and relatives). Number of alleles ranged from 1 to 4 with an average of two alleles per SSR locus. Mean PIC, observed heterozygosity and Shannon information index were found to be 0.258, 0.37 and 0.476 which suggests ncRNA-SSRs show higher polymorphism compared to genic SSRs but lower polymorphism compared to genomic SSRs. Thirty-six ncRNA-SSRs showed transferability ranging from 42.1% to 100%. Average genetic dissimilarity among wheat genotypes was found to be 0.29 based on Jaccard's dissimilarity. This is the first report of ncRNA-SSRs in wheat which will be useful for molecular breeding and genetic improvement of wheat.

Cross-transferability of SSR markers developed in Rhododendron species of Himalaya.

Rhododendron is a genus of evergreen woody ornamental plants of northern hemisphere with strong cold resistance, attractive flowers and high altitude adaptation capacity. The genus originated and diversified from Sino-Himalayan region and spread across the world, and has high species diversity in Northeast India. To assess cross-species amplification, we tested 32 microsatellites markers in fifteen taxa of the genus Rhododendron of North-eastern Himalaya, of which fourteen microsatellites were newly developed from Rhododendron simsii, and eighteen microsatellites were previously developed from Rhododendron catawbiense and Rhododendron mucronatum var. ripense. Nine pairs of primers were amplified successfully in all species, however, none of them was failed for amplification in any of the species. The average observed heterozygosity, expected heterozygosity and PIC value were recorded as 0.310, 0.433 and 0.379 respectively. Clustering based on neighbour-joining analysis revealed the potential of these markers to segregate species according to their subgenus level, however, subspecies exhibited closeness with each other. Cross-application of these microsatellite loci will provide a potentially useful tool to investigate the genetic diversity, population structure, gene flow, phylogenetics and evolutionary relationships in species of genus Rhododendron.

Development and characterization of bZIP transcription factor based SSRs in wheat.

Assessment of existing diversity is the key for germplasm conservation and crop improvement. Wheat (Triticum aestivum L.) is among the most important cereal crop and consumed by two billion world's populations. DNA-based markers are predominantly used for diversity characterization because they are easy to develop and not influenced by environment. Among them microsatellites (simple sequence repeats, SSRs) are most suitable due to their genome-wide distribution, hypervariability and reproducibility for their applications in diversity, genetic improvement, and molecular breeding. bZIP transcription factors play major roles in plants in light and stress signalling, seed development, and defence. A total of 846 SSRs were identified from 370 wheat cDNA sequences and a sub-set of 35 polymorphic TabZIPMS (TriticumaestivumbZIP MicroSatellites) was used for diversity and genetic structure analysis of 92 Indian wheat varieties and related species. 114 SSR variants ranging from 2 to 5 per SSR locus were detected for 35 SSRs in the varieties. Average polymorphic information content (PIC) and observed heterozygosity was found to be 0.135 and 0.838, respectively. Thirty-four SSRs showed cross-transferability into different related species. Combined Bayesian model and Jaccard's similarity based genetic clustering analysis revealed two clusters of 80 bread wheat varieties and one separate cluster of related species. In this study, a total 35 novel bZIP-derived SSRs were identified in a set 370 bZIP genes and shown high polymorphism and cross-species transferability in wheat. The findings provide resources for future utilization in genetic resource conservation, trait introgression, breeding and varietal development.

Identification and characterization of long non-coding RNAs regulating resistant starch biosynthesis in bread wheat (Triticum aestivum L.).

Resistant starch (RS) also known as healthy starch has shown several health benefits. Enhancing the RS through genetic modification approaches has huge commercial importance. Regulatory RNA like long non-coding RNA (lncRNA) plays an important role in gene regulation. In this study, we mined 63 transcriptome datasets of wheat belonging to 35 genotypes representing two seed developmental stages. Contrasting expression of a subset of lncRNAs in RS mutant lines compared to parent wheat variety 'C 306' signifies their probable role in RS biosynthesis. Further, lncRNA- TCONS_00130663 showed strong positive correlation (r2 = 1) with LYPL gene and strong negative correlation with SBEIIb (r2 = -0.94). We found TCONS_00130663 as positive regulator of LYPL gene through interaction with miR1128. Based on relative expression, in silico interaction and DSC analysis we hypothesize the dual role of TCONS_00130663 in RS type 2 and type 5. The study provides a useful resource for functional mechanism of lncRNAs.

Biolistic Delivery of Programmable Nuclease (CRISPR/Cas9) in Bread Wheat.

The discovery of site-specific programmable nucleases has led to a major breakthrough in the area of genome editing. In the past few years, CRISPR/Cas system has been utilized for genome editing of a large number of crops including cereals like wheat, rice, maize, and barley. In terms of consumption, wheat is second only to rice as the most important crop of the world. In the present chapter, we describe biolistic delivery method of ribonucleoprotein (RNP) complexes of programmable nuclease (CRISPR/Cas9) for targeted genome editing and selection-free screening of transformants in wheat. The method not only overcomes the problem of random integration into the genome but also reduces the off-targets. Besides the step-by-step protocol, plausible challenges and ways to overcome them are also discussed. By using the described method of biolistic delivery of CRISPR/Cas9 in plant systems, genome-edited plants can be identified within 11 weeks.

Novel intron length polymorphic (ILP) markers from starch biosynthesis genes reveal genetic relationships in Indian wheat varieties and related species.

Introns experience lesser selection pressure, thus are liable for higher polymorphism. Intron Length Polymorphic (ILP) markers designed from exon-flanking introns exploits this polymorphic potential and have been proved to be a robust co-dominant marker in eukaryotes. Wheat is among the most consumed cereal crop by majority of the word population. It is a rich source of calories in the form of stored starch. In the current study, starch biosynthesis genes were mined for development of ILP markers and their subsequent utilization for genetic characterization of popular Indian wheat varieties and transferability to wild relatives. Sixty-one markers generated 122 alleles and showed 77-88.5% transferability (mean PIC: 0.36) to the related species. A subset of markers showed clear genetic distinctions (Avg. genetic dissimilarity = 0.42) among Indian wheat varieties, signifying the importance of novel ILPs. 'Kenphad25' showed maximum genetic dissimilarity with 'K 8962' (0.82), while maximum genetic similarity was observed between 'Safed Lerma' and 'RAJ 4037' (0.1). This is the first report of ILP markers in wheat and will be a useful genomic resource for future germplasm conservation and molecular breeding studies.

Sound as a stimulus in associative learning for heat stress in Arabidopsis.

Plants are analogous to animals by responding physiologically and phenotypically to environmental changes. Until recently, the meaning of sound in the plant's life remains undiscovered. In this study, we investigated the role of music in response to heat stress and its application in memory and associative learning for stress tolerance in Arabidopsis. Significant upregulation of heat-responsive genes (HSFA3, SMXL7, and ATHSP101) in response to music suggests music has an advantage during heat stress. Moreover, the defensive conditioning experiment showed that plant learns to associate music with stress (heat) and elicit better response compared to music alone. Two heat-responsive genes, HSFA3 and ATCTL1, which are well known for their interaction and regulation of an array of heat shock proteins were found to play a key role in associative learning for heat stress in Arabidopsis. Our experiment highlights the application of sound in plant conditioning and as a stress reliever. Nonetheless, the persistence of memory awaits further experiments. We foresee the potential of artificial sound as an environment-friendly stimulus in conditioning the crops for upcoming stresses and reduce the yield loss, as an alternative to breeding and genetic modifications.

Discovery and Utilization of EST-SSR Marker Resource for Genetic Diversity and Population Structure Analyses of a Subtropical Bamboo, Dendrocalamus hamiltonii.

Dendrocalamus hamiltonii is a giant bamboo species native to Indian subcontinent with high economic importance. Nevertheless, highly outcross nature and flowering once in decades impose severe limitation in its propagation. Identification and mixed cultivation of genetically diverse genotypes may assist successful breeding and natural recombination of desirable traits. Characterization of existing genetic diversity and population structure are indispensable for efficient implementation of such strategies, which is facing a major challenge due to non-availability of sequence-based markers for the species. In this study, 8121 EST-SSR markers were mined from D. hamiltonii transcriptome data. Among all, tri-repeats were most represented (52%), with the abundance of CCG/CGG repeat motif. A set of 114 polymorphic markers encompassing epigenetic regulators, transcription factors, cell cycle regulators, signaling, and cell wall biogenesis, detected polymorphism and interaction (in silico) with important genes, that might have role in bamboo growth and development. Genetic diversity and population structure of the three D. hamiltonii populations (72 individuals) revealed moderate to high-level genetic diversity (mean alleles per locus: 5.8; mean PIC: 0.44) using neutral EST-SSR markers. AMOVA analysis suggests maximum diversity (59%) exists within population. High genetic differentiation (Gst = 0.338) and low gene flow (Nm = 0.49) were evident among populations. Further, PCoA, dendrogram, and Bayesian STRUCTURE analysis clustered three populations into two major groups based on geographical separations. In future, SSR marker resources created can be used for systematic breeding and implementation of conservation plans for sustainable utilization of bamboo complex.

Global Transcriptional Insights of Pollen-Pistil Interactions Commencing Self-Incompatibility and Fertilization in Tea [Camellia sinensis (L.) O. Kuntze].

This study explicates molecular insights commencing Self-Incompatibility (SI) and CC (cross-compatibility/fertilization) in self (SP) and cross (CP) pollinated pistils of tea. The fluorescence microscopy analysis revealed ceased/deviated pollen tubes in SP, while successful fertilization occurred in CP at 48 HAP. Global transcriptome sequencing of SP and CP pistils generated 109.7 million reads with overall 77.9% mapping rate to draft tea genome. Furthermore, concatenated de novo assembly resulted into 48,163 transcripts. Functional annotations and enrichment analysis (KEGG & GO) resulted into 3793 differentially expressed genes (DEGs). Among these, de novo and reference-based expression analysis identified 195 DEGs involved in pollen-pistil interaction. Interestingly, the presence of 182 genes [PT germination & elongation (67), S-locus (11), fertilization (43), disease resistance protein (30) and abscission (31)] in a major hub of the protein-protein interactome network suggests a complex signaling cascade commencing SI/CC. Furthermore, tissue-specific qRT-PCR analysis affirmed the localized expression of 42 DE putative key candidates in stigma-style and ovary, and suggested that LSI initiated in style and was sustained up to ovary with the active involvement of csRNS, SRKs & SKIPs during SP. Nonetheless, COBL10, RALF, FERONIA-rlk, LLG and MAPKs were possibly facilitating fertilization. The current study comprehensively unravels molecular insights of phase-specific pollen-pistil interaction during SI and fertilization, which can be utilized to enhance breeding efficiency and genetic improvement in tea.

Spatial transcriptome analysis provides insights of key gene(s) involved in steroidal saponin biosynthesis in medicinally important herb Trillium govanianum.

Trillium govanianum, an endangered medicinal herb native to the Himalaya, is less studied at the molecular level due to the non-availability of genomic resources. To facilitate the basic understanding of the key genes and regulatory mechanism of pharmaceutically important biosynthesis pathways, first spatial transcriptome sequencing of T. govanianum was performed. 151,622,376 (~11.5 Gb) high quality reads obtained using paired-end Illumina sequencing were de novo assembled into 69,174 transcripts. Functional annotation with multiple public databases identified array of genes involved in steroidal saponin biosynthesis and other secondary metabolite pathways including brassinosteroid, carotenoid, diterpenoid, flavonoid, phenylpropanoid, steroid and terpenoid backbone biosynthesis, and important TF families (bHLH, MYB related, NAC, FAR1, bZIP, B3 and WRKY). Differentially expressed large number of transcripts, together with CYPs and UGTs suggests involvement of these candidates in tissue specific expression. Combined transcriptome and expression analysis revealed that leaf and fruit tissues are the main site of steroidal saponin biosynthesis. In conclusion, comprehensive genomic dataset created in the current study will serve as a resource for identification of potential candidates for genetic manipulation of targeted bioactive metabolites and also contribute for development of functionally relevant molecular marker resource to expedite molecular breeding and conservation efforts in T. govanianum.

Genome-Wide Transcriptional Profiling to Elucidate Key Candidates Involved in Bud Burst and Rattling Growth in a Subtropical Bamboo (Dendrocalamus hamiltonii).

Bamboo, one of the fastest growing plants, can be a promising model system to understand growth. The study provides an insight into the complex interplay between environmental signaling and cellular machineries governing initiation and persistence of growth in a subtropical bamboo (Dendrocalamus hamiltonii). Phenological and spatio-temporal transcriptome analysis of rhizome and shoot during the major vegetative developmental transitions of D. hamiltonii was performed to dissect factors governing growth. Our work signifies the role of environmental cues, predominantly rainfall, decreasing day length, and high humidity for activating dormant bud to produce new shoot, possibly through complex molecular interactions among phosphatidylinositol, calcium signaling pathways, phytohormones, circadian rhythm, and humidity responses. We found the coordinated regulation of auxin, cytokinin, brassinosteroid signaling and cell cycle modulators; facilitating cell proliferation, cell expansion, and cell wall biogenesis supporting persistent growth of emerging shoot. Putative master regulators among these candidates were identified using predetermined Arabidopsis thaliana protein-protein interaction network. We got clues that the growth signaling begins far back in rhizome even before it emerges out as new shoot. Putative growth candidates identified in our study can serve in devising strategies to engineer bamboos and timber trees with enhanced growth and biomass potentials.