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.

Autophagy: a game changer for plant development and crop improvement.

MAIN CONCLUSION: Manipulation of autophagic pathway represents a tremendous opportunity for designing climate-smart crops with improved yield and better adaptability to changing environment. For exploiting autophagy to its full potential, identification and comprehensive characterization of adapters/receptor complex and elucidation of its regulatory network in crop plants is highly warranted.  Autophagy is a major intracellular trafficking pathway in eukaryotes involved in vacuolar degradation of cytoplasmic constituents, mis-folded proteins, and defective organelles. Under optimum conditions, autophagy operates at a basal level to maintain cellular homeostasis, but under stressed conditions, it is induced further to provide temporal stress relief. Our understanding of this highly dynamic process has evolved exponentially in the past few years with special reference to several plant-specific roles of autophagy. Here, we review the most recent advances in the field of autophagy in plants and discuss its potential implications in designing crops with improved stress and disease-tolerance, enhanced yield potential, and improved capabilities for producing metabolites of high economic value. We also assess the current knowledge gaps and the possible strategies to develop a robust module for biotechnological application of autophagy to enhance bioeconomy and sustainability of agriculture.

Phytohormones: Key players in the modulation of heavy metal stress tolerance in plants.

Heavy metal (HM) stress in plants has received considerable global attention as it threatens sustainable growth in agriculture worldwide. Hence, desperate efforts have been undertaken for combating the effects of this stress in plants. Interestingly, the use of phytohormones in reducing the impact of HM toxicity has gained much momentum in the recent past. Phytohormones act as chemical messengers that improve the HM stress resistance in plants, thus allowing them to retain their growth and developmental plasticity. Their exogenous application as well as manipulation of endogenous levels through precise targeting of their biosynthesis/signaling components is a promising approach for providing a protective shield against HM stress in plants. However, for the successful use of phytohormones for field plants exposed to HM toxicity, in-depth knowledge of the key pathways regulated by them is of prime importance. Hence, the present review mainly summarizes the key conceptual developments on the involvement of phytohormones in the mitigation of HM stress in plants. The role of various genes, proteins, and signaling components involved in phytohormones associated HM stress tolerance and their modulation has also been discussed. Thus, this update will pave the way for improving HM stress tolerance in plants with the advent of phytohormones for sustainable agriculture growth in the future.

In silico insights on diverse interacting partners and phosphorylation sites of respiratory burst oxidase homolog (Rbohs) gene families from Arabidopsis and rice.

BACKGROUND: NADPH oxidase (Nox) is a critical enzyme involved in the generation of apoplastic superoxide (O2-), a type of reactive oxygen species (ROS) and hence regulate a wide range of biological functions in many organisms. Plant Noxes are the homologs of the catalytic subunit from mammalian NADPH oxidases and are known as respiratory burst oxidase homologs (Rbohs). Previous studies have highlighted their versatile roles in tackling different kind of stresses and in plant growth and development. In the current study, potential interacting partners and phosphorylation sites were predicted for Rboh proteins from two model species (10 Rbohs from Arabidopsis thaliana and 9 from Oryza sativa japonica). The present work is the first step towards in silico prediction of interacting partners and phosphorylation sites for Rboh proteins from two plant species. RESULTS: In this work, an extensive range of potential partners (unique and common), leading to diverse functions were revealed from interaction networks and gene ontology classifications, where majority of AtRbohs and OsRbohs play role in stress-related activities, followed by cellular development. Further, 68 and 38 potential phosphorylation sites were identified in AtRbohs and OsRbohs, respectively. Their distribution, location and kinase specificities were also predicted and correlated with experimental data as well as verified with the other EF-hand containing proteins within both genomes. CONCLUSIONS: Analysis of regulatory mechanisms including interaction with diverse partners and post-translational modifications like phosphorylation have provided insights regarding functional multiplicity of Rbohs. The bioinformatics-based workflow in the current study can be used to get insights for interacting partners and phosphorylation sites from Rbohs of other plant species.

Reactive oxygen species dynamics in roots of salt sensitive and salt tolerant cultivars of rice.

Salinity stress is one of the major constraints for growth and survival of plants that affects rice productivity worldwide. Hence, in the present study, roots of two contrasting salinity sensitive cultivars, IR64 (IR64, salt sensitive) and Luna Suvarna (LS, salt tolerant) were compared with regard to the levels of reactive oxygen species (ROS) to derive clues for their differential salt stress adaptation mechanisms. In our investigation, the tolerant cultivar exhibited longer primary roots, more lateral roots, higher root number leading to increased root biomass, with respect to IR64. It was observed that LS roots maintained higher level of H2O2 in comparison to IR64. The activities of various enzymes involved in enzymatic antioxidant defense mechanism (SOD, CAT, GPX, DHAR and MDHAR) were found to be greater in LS roots. Further, the higher transcript level accumulation of genes encoding ROS generating (RbohA, RbohD and RbohE) and scavenging enzymes (Fe-SOD, Chloroplastic Cu/Zn-SOD, CAT and DHAR) were noticed in the roots of tolerant cultivar, LS. Moreover, the content of other stress markers such as total protein and proline were also elevated in LS roots. While, the expression of proline biosynthesis gene (P5CS) and proline catabolism gene (PDH) was observed to be lower in LS.

Structural complexity and functional diversity of plant NADPH oxidases.

Plant NADPH oxidases also known as respiratory burst oxidase homologs (Rbohs) are a family of membrane-bound enzymes that play diverse roles in the defense response and morphogenetic processes via regulated generation of reactive oxygen species. Rbohs are associated with a variety of functions, although the reason for this is not clear. To evaluate using bioinformatics, the possible mechanisms for the observed functional diversity within the plant kingdom, 127 Rboh protein sequences representing 26 plant species were analyzed. Multiple clusters were identified with gene duplications that were both dicot as well as monocot-specific. The N-terminal sequences were observed to be highly variable. The conserved cysteine (equivalent of Cys890) in C-terminal of AtRbohD suggested that the redox-based modification like S-nitrosylation may regulate the activity of other Rbohs. Three-dimensional models corresponding to the N-terminal domain for Rbohs from Arabidopsis thaliana and Oryza sativa were constructed and molecular dynamics studies were carried out to study the role of Ca2+ in the folding of Rboh proteins. Certain mutations indicated possibly affect the structure and function of the plant NADPH oxidases, thereby providing the rationale for further experimental validation.

Interdependency of Reactive Oxygen Species generating and scavenging system in salt sensitive and salt tolerant cultivars of rice.

BACKGROUND: Salinity stress is a major constrain in the global rice production and hence serious efforts are being undertaken towards deciphering its remedial strategies. The comparative analysis of differential response of salt sensitive and salt tolerant lines is a judicious approach to obtain essential clues towards understanding the acquisition of salinity tolerance in rice plants. However, adaptation to salt stress is a fairly complex process and operates through different mechanisms. Among various mechanisms involved, the reactive oxygen species mediated salinity tolerance is believed to be critical as it evokes cascade of responses related to stress tolerance. In this background, the present paper for the first time evaluates the ROS generating and the scavenging system in tandem in both salt sensitive and salt tolerant cultivars of rice for getting better insight into salinity stress adaptation. RESULTS: Comparative analysis of ROS indicates the higher level of hydrogen peroxide (H2O2) and lower level of superoxide ions (O(2-)) in the salt tolerant as compared to salt sensitive cultivars. Specific activity of ROS generating enzyme, NADPH oxidase was also found to be more in the tolerant cultivars. Further, activities of various enzymes involved in enzymatic and non enzymatic antioxidant defence system were mostly higher in tolerant cultivars. The transcript level analysis of antioxidant enzymes were in alignment with the enzymatic activity. Other stress markers like proline were observed to be higher in tolerant varieties whereas, the level of malondialdehyde (MDA) equivalents and chlorophyll content were estimated to be more in sensitive. CONCLUSION: The present study showed significant differences in the level of ROS production and antioxidant enzymes activities among sensitive and tolerant cultivars, suggesting their possible role in providing natural salt tolerance to selected cultivars of rice. Our study demonstrates that the cellular machinery for ROS production and scavenging system works in an interdependent manner to offer better salt stress adaptation in rice. The present work further highlights that the elevated level of H2O2 which is considered as a key determinant for conferring salt stress tolerance to rice might have originated through an alternative route of photocatalytic activity of chlorophyll.

OsMADS26 Negatively Regulates Resistance to Pathogens and Drought Tolerance in Rice.

Functional analyses of MADS-box transcription factors in plants have unraveled their role in major developmental programs (e.g. flowering and floral organ identity) as well as stress-related developmental processes, such as abscission, fruit ripening, and senescence. Overexpression of the rice (Oryza sativa) MADS26 gene in rice has revealed a possible function related to stress response. Here, we show that OsMADS26-down-regulated plants exhibit enhanced resistance against two major rice pathogens: Magnaporthe oryzae and Xanthomonas oryzae. Despite this enhanced resistance to biotic stresses, OsMADS26-down-regulated plants also displayed enhanced tolerance to water deficit. These phenotypes were observed in both controlled and field conditions. Interestingly, alteration of OsMADS26 expression does not have a strong impact on plant development. Gene expression profiling revealed that a majority of genes misregulated in overexpresser and down-regulated OsMADS26 lines compared with control plants are associated to biotic or abiotic stress response. Altogether, our data indicate that OsMADS26 acts as an upstream regulator of stress-associated genes and thereby, a hub to modulate the response to various stresses in the rice plant.

High throughput characterizations of poly(A) site choice in plants.

The polyadenylation of mRNA in eukaryotes is an important biological process. In recent years, significant progress has been made in the field of mRNA polyadenylation owing to the advent of the next generation DNA sequencing technologies. The high-throughput sequencing capabilities have resulted in the direct experimental determinations of large numbers of polyadenylation sites, analysis of which has revealed a vast potential for the regulation of gene expression in eukaryotes. These collections have been generated using specialized sequencing methods that are targeted to the junction of 3'-UTR and the poly(A) tail. Here we present three variations of such a protocol that has been used for the analysis of alternative polyadenylation in plants. While all these methods use oligo-dT as an anchor to the 3'-end, they differ in the means of generating an anchor for the 5'-end in order to produce PCR products suitable for effective Illumina sequencing; the use of different methods to append 5' adapters expands the possible utility of these approaches. These methods are versatile, reproducible, and may be used for gene expression analysis as well as global determinations of poly(A) site choice.

Biotechnological interventions in Withania somnifera (L.) Dunal.

Withania somnifera is one of the most valued plants and is extensively used in Indian, Unani, and African systems of traditional medicine. It possess a wide array of therapeutic properties including anti-arthritic, anti-aging, anti-cancer, anti-inflammatory, immunoregulatory, chemoprotective, cardioprotective, and recovery from neurodegenerative disorders. With the growing realization of benefits and associated challenges in the improvement of W. somnifera, studies on exploration of genetic and chemotypic variations, identification and characterization of important genes, and understanding the secondary metabolites production and their modulation has gained significant momentum. In recent years, several in vitro and in vivo preclinical studies have facilitated the validation of therapeutic potential of the phytochemicals derived from W. somnifera and have provided necessary impetus for gaining deeper insight into the mechanistic aspects involved in the mode of action of these important pharmaceutically active constituents. The present review highlights some of the current developments and future prospects of biotechnological intervention in this important medicinal plant.

Retron Library Recombineering: Next Powerful Tool for Genome Editing after CRISPR/Cas.

Retron library recombineering (RLR) is a powerful tool in the field of genome editing that exceeds the scope and specificity of the CRISPR/Cas technique. In RLR, single-stranded DNA produced in vivo by harnessing the in-built potential of bacterial retrons is used for replication-dependent genome editing. RLR introduces several genomic variations at once, resulting in pooled and barcoded variant libraries, thus permitting multiplexed applications. Retron-generated RT-DNA has already shown promise for use in genome editing. Thus, this new tool will result in fresh, intriguing, and surprising developments in molecular biology and its juncture with other disciplines of research, including medicine, agriculture, and microbiology. In this review, we discuss the current state of this brand-new tool that could eventually boost genome editing research.

Auxin: a master regulator in plant root development.

The demand for increased crop productivity and the predicted challenges related to plant survival under adverse environmental conditions have renewed the interest in research in root biology. Various physiological and genetic studies have provided ample evidence in support of the role of plant growth regulators in root development. The biosynthesis and transport of auxin and its signaling play a crucial role in controlling root growth and development. The univocal role of auxin in root development has established it as a master regulator. Other plant hormones, such as cytokinins, brassinosteroids, ethylene, abscisic acid, gibberellins, jasmonic acid, polyamines and strigolactones interact either synergistically or antagonistically with auxin to trigger cascades of events leading to root morphogenesis and development. In recent years, the availability of biological resources, development of modern tools and experimental approaches have led to the advancement of knowledge in root development. Research in the areas of hormone signal perception, understanding network of events involved in hormone action and the transport of plant hormones has added a new dimension to root biology. The present review highlights some of the important conceptual developments in the interplay of auxin and other plant hormones and associated downstream events affecting root development.

Physiological and molecular insight of microbial biostimulants for sustainable agriculture.

Increased food production to cater the need of growing population is one of the major global challenges. Currently, agro-productivity is under threat due to shrinking arable land, increased anthropogenic activities and changes in the climate leading to frequent flash floods, prolonged droughts and sudden fluctuation of temperature. Further, warm climatic conditions increase disease and pest incidences, ultimately reducing crop yield. Hence, collaborated global efforts are required to adopt environmentally safe and sustainable agro practices to boost crop growth and productivity. Biostimulants appear as a promising means to improve growth of plants even under stressful conditions. Among various categories of biostimulants, microbial biostimulants are composed of microorganisms such as plant growth-promoting rhizobacteria (PGPR) and/or microbes which stimulate nutrient uptake, produce secondary metabolites, siderophores, hormones and organic acids, participate in nitrogen fixation, imparts stress tolerance, enhance crop quality and yield when applied to the plants. Though numerous studies convincingly elucidate the positive effects of PGPR-based biostimulants on plants, yet information is meagre regarding the mechanism of action and the key signaling pathways (plant hormone modulations, expression of pathogenesis-related proteins, antioxidants, osmolytes etc.) triggered by these biostimulants in plants. Hence, the present review focuses on the molecular pathways activated by PGPR based biostimulants in plants facing abiotic and biotic challenges. The review also analyses the common mechanisms modulated by these biostimulants in plants to combat abiotic and biotic stresses. Further, the review highlights the traits that have been modified through transgenic approach leading to physiological responses akin to the application of PGPR in the target plants.

Plant lectins: Classical molecules with emerging roles in stress tolerance.

Biotic and abiotic stresses impose adverse effects on plant's development, growth, and production. For the past many years, researchers are trying to understand the stress induced responses in plants and decipher strategies to produce stress tolerant crops. It has been demonstrated that molecular networks encompassing an array of genes and functional proteins play a key role in generating responses to combat different stresses. Newly, there has been a resurgence of interest to explore the role of lectins in modulating various biological responses in plants. Lectins are naturally occurring proteins that form reversible linkages with their respective glycoconjugates. To date, several plant lectins have been recognized and functionally characterized. However, their involvement in stress tolerance is yet to be comprehensively analyzed in greater detail. The availability of biological resources, modern experimental tools, and assay systems has provided a fresh impetus for plant lectin research. Against this backdrop, the present review provides background information on plant lectins and recent knowledge on their crosstalks with other regulatory mechanisms, which play a remarkable role in plant stress amelioration. It also highlights their versatile role and suggests that adding more information to this under-explored area will usher in a new era of crop improvement.

Mitigation of adverse effects of chlorpyrifos by 24-epibrassinolide and analysis of stress markers in a rice variety Pusa Basmati-1.

The present paper first time reports the role of 24-epibrassinolide (EBL) in mitigating the adverse effects of Chlorpyrifos (CPF), a broad spectrum organophosphate insecticide by regulating the antioxidant defence system in an elite indica rice variety Pusa Basmati-1. It investigates the effect of CPF (0.02%, 0.04% and 0.06%) and EBL (10(-11), 10(-9) and 10(-7)M) treatments alone and in combination on rice seedlings. Various growth parameters, protein, proline and malondialdehyde (MDA) content and activities of antioxidant enzymes of seedlings were analysed. CPF showed an adverse effect on growth and protein content of seedlings whereas it leads to an enhancement in the level of MDA and proline content. The activities of antioxidant enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), guaiacol peroxidase (GPX) and monodehydroascorbate reductase (MDHAR) increased after treatment with CPF. Application of different concentrations of EBL along with CPF resulted in an overall improvement in the growth, level of protein and proline content and in the activity of various antioxidant enzymes whereas a decline in the levels of MDA content was observed. The work also investigated the changes at the transcript level of some key antioxidant enzymes like Cu/Zn-SOD, Fe-SOD, Mn-SOD, APX, CAT and GR. The expression of most of the genes was enhanced in response to CPF treatment. Application of EBL in conjunction with CPF resulted in a distinct enhancement in the transcript accumulation of Fe-SOD and CAT showing their important role in EBL mediated amelioration of CPF induced stress.

Genome Editing: A Promising Approach for Achieving Abiotic Stress Tolerance in Plants.

The susceptibility of crop plants towards abiotic stresses is highly threatening to assure global food security as it results in almost 50% annual yield loss. To address this issue, several strategies like plant breeding and genetic engineering have been used by researchers from time to time. However, these approaches are not sufficient to ensure stress resilience due to the complexity associated with the inheritance of abiotic stress adaptive traits. Thus, researchers were prompted to develop novel techniques with high precision that can address the challenges connected to the previous strategies. Genome editing is the latest approach that is in the limelight for improving the stress tolerance of plants. It has revolutionized crop research due to its versatility and precision. The present review is an update on the different genome editing tools used for crop improvement so far and the various challenges associated with them. It also highlights the emerging potential of genome editing for developing abiotic stress-resilient crops.

Critical factors influencing in vitro propagation and modulation of important secondary metabolites in Withania somnifera (L.) dunal.

Withania somnifera (L.) Dunal is a valuable medicinal plant in the Solanaceae family. It is commonly known as Ashwagandha and is widely distributed around the globe. It has multiple pharmacological properties owing to the existence of diverse secondary metabolites viz., withanolide A, withanolide D, withaferin A, and withanone. It is in great demand in the herbal industry because of its extensive use. In this background, the major challenge lies in the rapid multiplication of elite cultivars of W. somnifera in order to produce genetically and phytoconstituents uniform plant material for pharmaceutical industries. Thus it is necessary to explore various biotechnological approaches for the clonal mass propagation and synthesis of pharmaceutically important constituents in W. somnifera. Though there are several studies on in vitro propagation on W. somnifera, yet many factors that critically influence the in vitro response and withanolides production need to be fine-tuned in the pretext of the existing knowledge. The current review focuses on the advancements and prospects in biotechnological interventions to meet the worldwide demands for W. somnifera and its bioactive compounds. This update on in vitro studies on W. somnifera will be useful to many researchers, entrepreneurs, and herbal industries looking for its in vitro mass multiplication and scientific utilization.

Biotechnological interventions in banana: current knowledge and future prospects.

Banana is an important food crop responsible for ensuring food security, nutrition, and employment for a significant portion of the world population. It has fairly broad genetic diversity and is distributed widely across the globe. Due to its socio-economic importance, there has been growing demand for healthy and improved planting materials of banana. In recent years many companies and organizations are working hard to narrow down the gap between demand and supply of quality planting materials. The other challenges includes its susceptibility to adverse environmental conditions, attack of various pests/pathogens and improvement of nutritional quality of bananas. To address these issues, refinement of existing techniques and introduction of new experimental tools are required. However, the genetic improvement of bananas to a large extent is limited by using conventional methods due to polyploidy, heterozygosity, and sterility of this plant. For rapid multiplication and obtaining disease free and healthy plants, efficient in vitro propagation techniques and fine tuning of the existing protocols are being tried in many laboratories across the globe. Besides, for developing a successful protocol for propagation of different cultivars of bananas, a deeper understanding of the factors associated with various steps of its multiplication till transfer to the land is immensely critical. Similarly, developing biotic and abiotic stress tolerant banana and enhancing its commercial value through biotechnological interventions could be very useful. The key intent of this review is to highlight the research endeavor in this direction, associated challenges and future prospects.

OsSalT gene cloned from rice provides evidence of its role in salinity and drought stress tolerance.

Abiotic stresses impose a huge threat to agricultural productivity and global food security. To counter this challenge, the precise identification of the right candidate gene (s) for conferring abiotic stress tolerance without compromising the growth and yield is crucial. OsSalT is identified as a salt stress responsive gene located on SalTol QTL of chromosome 1 of rice, however, there is no genetic evidence of its function and probable pathway of its regulation. To get better insights into its functioning, earlier we elucidated the structure of SALT protein at atomic scale {PDB ID (5GVY)} and solution state that provided key clues on the probable mode of its action. Herein, we report the modulation of OsSalT gene in response to various factors and its functional characterization. Results indicate that OsSalT operates through both abscisic acid and gibberellic acid-dependent pathways and is linked to the adaptive stress mechanisms of plants. Its overexpression in a model plant resulted in improved salinity and drought stress tolerance. The OsSalT transformed plants also showed vigorous root growth, early flowering, and better seed germination. The triggering of multiple responses by OsSalT suggested that modulation of such mannose-binding lectin could be a potential game-changer for the improvement of many crops in future.

Effect of 28-homobrassinolide on antioxidant defence system in Raphanus sativus L. under chromium toxicity.

Heavy metals have emerged as major environmental contaminants due to rapid industrialization and urbanization. The genotoxic, mutagenic and carcinogenic effects of heavy metal like chromium (Cr) on man, animals and plants have been documented. In plants, accumulation of heavy metals beyond critical levels generates oxidative stress. This stress is generally overcome by antioxidant defence system and stress shielding phytohormones. Thus, the present study has been focused to analyze the effect of one of imperative group of plant hormones, i.e., brassinosteroids (BRs) which have been reported for its protective properties for wide array of environmental stresses. Raphanus sativus L. (Pusa Chetaki) seeds pre-treated with different concentrations of 28-homobrassinolide (28-HBL) were raised under various concentrations of Cr(VI). It was observed that 28-HBL treatment considerably reduced the impact of Cr-stress on seedlings which was evinced upon analysis of morphological and biochemical parameters of 7-days old radish seedlings. The toxic effects of Cr in terms of reduced growth, lowered contents of chlorophyll (Chl), protein, proline; increased malondialdehyde (MDA) content and elevated metal uptake were ameliorated by applications of 28-HBL. Also, the activities of all the antioxidant enzymes except guaiacol peroxidase (POD), increased significantly when subjected to Cr stress in combination with 28-HBL. Overall, seed pre-soaking treatment of 28-HBL at 10(-7) M was most effective in ameliorating Cr stress. The present work emphasizes the protective role of 28-HBL on regulation of antioxidant enzymes and its possible link in amelioration of stress in plants.