The overall analysis of the results in terms of the influence of the three tested endophytes on the biosynthetic potentials of the normal and composite plants in their leaves and roots pertaining to the three desired withanolides revealed very interesting outcomes which highlights the future applicability of this study. The WPR17 endophyte demonstrated the second optimum efficiency which also boosted the foliar production trend of these two above-stated withanolides WFA and WLA in both the control and composite plants, where the latter superseded the former by Expression of all studied genes in roots of in-vitro normal plants was increased by selected endophyte inoculation Fig.
Similarly, leaves and roots of endophytes inoculated in-vitro composite plants also had increased expression of studied pathway genes compared to non-inoculated endophyte-free control plants Fig. Effect of endophyte inoculation on the expression of withanolide biosynthesis genes in in-vitro grown normal and composite Withania somnifera plant. Expression of different withanolide biosynthesis genes was analyzed. Endophytes have promising potential for sustainable agriculture as they can promote plant growth and confer tolerance to plant from environmental stresses 31 , 32 , 33 , 34 , They are also the source of therapeutically important novel chemicals 36 , 37 , Earlier work from our laboratory has indicated that some endophytes may enhance the secondary metabolite production of the medicinal plants 39 , 40 , 42 , We have shown that medicinal plant opium poppy harbour numerous endophytes residing in different parts of the plants and play a role in a tissue-specific manner Endophytes associated with opium leaves were involved in improving photosynthetic efficiency of the plant while the endophytes associated with capsule major site for secondary metabolites production were involved in improving secondary metabolites specifically benzylisoquinoline alkaloid production No such relation was observed in Withania plants as far as shoot growth is concerned as the endophytes isolated both from leaf and root could enhance the shoot growth.
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However, endophytes isolated from roots could significantly enhance the root growth, supporting our earlier study suggesting their tissue specific roles Two fungal endophytes Curvularia sp. In the present study, we demonstrated an important role of endophytes in improving plant yield and secondary metabolite production in medicinally important W. We could isolate and identify a total of 29 bacterial endophytes and 11 fungal endophytes.
The role of these isolated endophytes was studied by inoculating them in endophyte-free plants and compared with the non-inoculated endophyte-free control plant. It was observed that few isolated fungal endophytes could improve the photosynthetic efficiency of W. Improved photosynthetic efficiency due to endophyte inoculation resulted in increased biomass of Withania plant that was evident from the higher shoot and root biomass.
As both leaves and roots are the economic parts, used for the extraction of withanolides, therefore, these fungal endophytes can be used for the substantial improvement of biomass. Improved growth in endophyte-inoculated Withania plants may be due to indole acetic acid production and phosphate solubilization activity of endophytes.
Withanolides are the major secondary metabolites responsible for pharmacological properties of different parts of W. Inoculation with endophytes modulated the content of withanolides in leaves and roots of W. Surprisingly, some endophytes induced the synthesis of withaferin A in roots 0. This clearly shows a strong involvement of endophytes in modulating the biosynthetic pathway.
Expression study of different genes involved in withanolide biosynthesis revealed the possible mechanism associated with endophyte mediated changes in withanolide biosynthesis.
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We observed that endophyte inoculation modulated the expression of genes of withanolide biosynthesis. Endophytes inoculation upregulated the expression of HMGR both in leaves and roots , involved in the biosynthesis of isopentenylnpyrophosphate IPP via MVA pathway which is the predominant pathway for biosynthesis of withanolides 48 , and this might be a key reason for the higher production of withanolides in endophyte-inoculated plants. It might be due to the constant association of these endophytes with roots their site of isolation that could specifically modulate their expression in roots.
The expression of DXS , DXR and FPPS remained unaffected in the leaves of endophyte inoculated plants, however, their expression was upregulated in roots and this might be a possible reason for induction of withaferin A synthesis in roots. Selected endophyte inoculation could increase the in planta content of IAA in leaves and roots that could enhance withanolide biosynthesis. Improvement of withaferin A production by application of nitrogen significantly upregulating structural and regulatory genes of withanolide biosynthesis also supports our present observation The previous report demonstrated that IAA favours the biosynthesis of withaferin A in in-vitro root cultures of W.
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Expression of SQS , encoding squalene synthase catalysing the condensation of farnesyl pyrophosphate FPP to produce squalene a key intermediate for biosynthesis of various triterpenoids was found to be upregulated in leaves and roots of all the selected endophyte-inoculated Withania plant. Overexpression of SQS in W. Overexpression of SQS in cell suspension culture of W.
Therefore, upregulated expression of SQS up to 3. Endophyte-inoculated plants had upregulated expression of SQE which encodes squalene epoxidase which is a rate limiting enzyme in the withanolide biosynthetic pathway 53 , Cascading influence of SQE on the upregulation of downstream genes and its genetic manipulation in W.
Therefore, higher expression of SQE up to 4.
Endophyte-inoculation could upregulate the expression of CAS which forms cycloartenol the precursor for withanolide biosynthesis and acts at metabolic branching point for synthesis of withanolides and various triterpenes such as saponins Fig. Previous reports show that CAS overexpression and silencing resulted in an increase and decrease of withanolide content respectively Few endophytes modulated the expression of CPRs which encode cytochrome P reductase. Ps are involved in the primary and secondary metabolism of a plant by catalysing various reaction such as hydroxylations, epoxidations, sulfoxidations, dealkylations, peroxidations, reductive dehalogenations and isomerisation reactions 57 , Endophytes also modulated the expression of SGT sterol glucosyltransferases responsible for glyco-transformations of steroids.
All the selected endophyte-inoculations upregulated the expression of HMGR in both leaf and root tissues, indicating that it may be a promising candidate gene that can be used for increasing in planta withanolide production. Previously, HMGR as rate limiting enzyme of isoprenoids biosynthesis of eukaryotes has also been suggested Piriformospora indica elicitation increased withaferin A biosynthesis by upregulating the regulatory genes of MEP, MVA and withanolide biosynthetic pathway and showed the highest expression of HMGR in cell suspension cultures of Withania somnifera WPR17 and WPSinoculation upregulated the expression of all the key genes of withanolide biosynthesis in roots.
Therefore, these are the most promising candidates that can be used for the improvement of in planta withanolide production. As roots of the W. The utilization of composite plants had earlier been reported as a successful alternative approach for the functional characterization of any targeted gene s in the host plants, where the transformation and regeneration are tedious and lengthy This technology has also been found competent for several other studies such as nutrient and hormone uptake, interactions with root nodulating bacteria and mycorrhizal symbiotic association The present study has first time explored the potentials of the composite plants with respect to evaluating the influence of external applications of targeted endophytes with the overall intention of amplifying the host-endophyte interaction interphase to best of our knowledge.
Here, we developed the composite plant of W. Normal plants were regenerated in-vitro from nodal explants, and composite plants were generated by inoculating wild-type A.
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Successful integration of rol gene in the genome of normal plants could result in the development of composite plants having more roots than the normal plants. Shoot and root regeneration in nodal explants was found to be similar as reported previously 61 , 62 , 63 , 64 , 65 , Furthermore, in general the inoculation with selected endophytes WPL16, WPL17 and WPS23 enhanced the content of all the measured withanolide WFA, DWL and WLA in the leaves and roots of the composite plants, which is due to the upregulation of the expression of key genes of withanolide biosynthesis as observed in the case of in-vivo normal plants.
Similarly presence of WFA and DWL in roots of composite plant which was not detected in normal plants in both endophyte inoculated and non-inoculated plants also indicates the change of flux of withanolide biosynthesis from leaves to roots, which might have resulted due to the Ri T-DNA mediated changed nature of the composite plant.
To summarize, results of the present study suggest that the medicinal plant W. These endophytes may play an important role in the biosynthesis of important secondary metabolites and also have the potential to improve plant shoot and root biomass by improving the photosynthetic pigments, photosynthesis rate, transpiration rate and stomatal conductance through growth promotion activities such as IAA production and phosphate solubilization. We also demonstrated that W.
The presence of few IAA-producing and nitrogen-fixing root-associated endophytes that can induce the production of leaf alkaloids withaferin A in roots by upregulating the expression of withanolide biosynthesis genes especially MEP pathway genes DXS and DXR as promising candidates for enhancing the withaferin A biosynthesis in Withania root is also suggested.
Application of these endophytes in Withania agriculture will benefit growers to harvest higher amounts of both withanolide A and withaferin A from roots. Although the mechanism associated with endophyte-mediated modulation of host plant metabolism is really difficult to unravel, their possible role in promoting plant growth and yields, and pharmaceutically important withanolide biosynthesis cannot be ignored. Formulation and testing of microbial consortia consisted of endophytes enhancing plant biomass and endophytes enhancing withanolide content may empower host plant to produce higher biomass coupled with more withanolide content.
Endophytes were isolated from healthy green leaves, root and seeds of field-grown Withania somnifera genotype Poshita plant following the procedure described previously 39 , Isolation of endophytes was done in triplicate. Samples were discarded if the growth was detected in the sterility check samples. A representative of each endophyte as distinct from their colony morphology was transferred to a fresh plate and pure culture was established.
Endophyte-free Withania plants were used to study the effects of treatment with isolated endophytes.
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Endophytes-free Withania plants were generated following previously established procedure 39 , No microbes were obtained on incubated plates. Therefore, these W.
These seedlings were uprooted delicately to minimize the damage to their roots and inoculated with isolated endophytes. The roots of non-inoculated endophyte-free control were dipped in PBS for the same duration. Plants were watered with sterile water as and when required. In all experimental analyses, endophyte-inoculated plants were compared with the non-inoculated endophyte-free control plants that developed from endophyte-free Withania seeds. Presence of inoculated endophytes in Withania plants was confirmed by re-isolating the endophytes from tissues of endophyte-inoculated plants before carrying out further experimental analyses.
Sampling for all analyses was carried out at the same stage 90 d which is an intermediate stage neither too young nor too old and position of leaf third leaf from top for minimizing the variations dependent on developmental stage of plant and other variables. In-vitro culture of W.
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Surface sterilization of explants was done with 0. The transformation experiments for the development of composite plants were carried out following previously published protocol 62 with slight modification utilizing the wild type of A4 strain of A. In this study, the needle-pricking method was applied at the cut end of nodal explant by inoculation of over-night grown bacterial suspension O.
For control plants, only PBS without endophyte was used. Plants were grown under confined glasshouse condition. DNA was extracted from the in-vitro raised transformed roots of composite plants CPR and normal plant root NPR according to the protocol described previously The PCR conditions were followed as described previously Shoot and root biomass of days old W. Leaves third from top and roots of days old Withania plant were taken for alkaloid extraction. In the case of in-vitro grown normal and composite Withania plant leaves and roots samples were taken at 60 d stage after glass house acclimatization.
Tissues were dried and ground to fine powder. The extraction process was repeated three times.