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The leaf-specific Catharanthus roseus alkaloid, vindoline, is the major bottleneck precursor in the production of scarce and costly anticancer bisindoles (vincristine and vinblastine). The final steps of its biosynthesis and storage occur in the laticifers. Earlier, we have shown that vindoline content is directly related to laticifer number. Pectin remodeling enzymes, like pectin methylesterase (PME), are known to be involved in laticifer development. A search in the croFGD yielded a leaf-abundant CrPME isoform that co-expressed with a few vindoline biosynthetic genes. Full-length cloning, tissue-specific expression profiling, and in silico analysis of CrPME were carried out. It was found to possess all the specific characteristics of a typical plant PME. Transient silencing (through VIGS) and overexpression of CrPME in C. roseus indicated a direct relationship between its expression and vindoline content. Comparative analysis of transcript abundance and enzyme activity in three familial C. roseus genotypes differing significantly in their vindoline content and laticifer count (CIM-Sushil > Dhawal > Nirmal) also corroborated the positive relationship of CrPME expression with vindoline content. This study highlights the possible role of CrPME, a cell wall remodeling enzyme, in modulating laticifer-associated secondary metabolism. [ABSTRACT FROM AUTHOR]

Plant lipocalins perform diverse functions. Recently, allene oxide cyclase, a lipocalin family member, has been shown to co‐express with vindoline pathway genes in Catharanthus roseus under various biotic/abiotic stresses. This brought focus to another family member, a temperature‐induced lipocalin (CrTIL), which was selected for full‐length cloning, tissue‐specific expression profiling, in silico characterization, and upstream genomic region analysis for cis‐regulatory elements. Stress‐mediated variations in CrTIL expression were reflected as disturbances in cell membrane integrity, assayed through measurement of electrolyte leakage and lipid peroxidation product, MDA, which implicated the role of CrTIL in maintaining cell membrane integrity. For ascertaining the function of CrTIL in maintaining membrane stability and elucidating the relationship between CrTIL expression and vindoline content, if any, a direct approach was adopted, whereby CrTIL was transiently silenced and overexpressed in C. roseus. CrTIL silencing and overexpression confirmed its role in the maintenance of membrane integrity and indicated an inverse relationship of its expression with vindoline content. GFP fusion‐based subcellular localization indicated membrane localization of CrTIL, which was in agreement with its role in maintaining membrane integrity. Altogether, the role of CrTIL in maintaining membrane structure has possible implications for the intracellular sequestration, storage, and viability of vindoline. [ABSTRACT FROM AUTHOR]

SUMMARY: In plants, geranylgeranyl diphosphate (GGPP, C20) synthesized by GGPP synthase (GGPPS) serves as precursor for vital metabolic branches including specialized metabolites. Here, we report the characterization of a GGPPS (CrGGPPS2) from the Madagascar periwinkle (Catharanthus roseus) and demonstrate its role in monoterpene (C10)‐indole alkaloids (MIA) biosynthesis. The expression of CrGGPPS2 was not induced in response to methyl jasmonate (MeJA), and was similar to the gene encoding type‐I protein geranylgeranyltransferase_β subunit (CrPGGT‐I_β), which modulates MIA formation in C. roseus cell cultures. Recombinant CrGGPPS2 exhibited a bona fide GGPPS activity by catalyzing the formation of GGPP as the sole product. Co‐localization of fluorescent protein fusions clearly showed CrGGPPS2 was targeted to plastids. Downregulation of CrGGPPS2 by virus‐induced gene silencing (VIGS) significantly decreased the expression of transcription factors and pathway genes related to MIA biosynthesis, resulting in reduced MIA. Chemical complementation of CrGGPPS2‐vigs leaves with geranylgeraniol (GGol, alcoholic form of GGPP) restored the negative effects of CrGGPPS2 silencing on MIA biosynthesis. In contrast to VIGS, transient and stable overexpression of CrGGPPS2 enhanced the MIA biosynthesis. Interestingly, VIGS and transgenic‐overexpression of CrGGPPS2 had no effect on the main GGPP‐derived metabolites, cholorophylls and carotenoids in C. roseus leaves. Moreover, silencing of CrPGGT‐I_β, similar to CrGGPPS2‐vigs, negatively affected the genes related to MIA biosynthesis resulting in reduced MIA. Overall, this study demonstrated that plastidial CrGGPPS2 plays an indirect but necessary role in MIA biosynthesis. We propose that CrGGPPS2 might be involved in providing GGPP for modifying proteins of the signaling pathway involved in MIA biosynthesis. Significance Statement: Our study reveals a link between C20 metabolic intermediate (GGPP) and (C10)‐monoterpene indole alkaloids (MIA), thus adding new insights into MIA formation in Catharanthus roseus. The findings demonstrate that the plastid‐localized bona fide CrGGPPS2 plays an important role in MIA biosynthesis through its potential involvement in providing the precursor for protein‐geranylgeranyl‐transferase (PGGT) involved in modulation of the MIA pathway [ABSTRACT FROM AUTHOR]

Transgenic Catharanthus roseus plants (transgenic Dhawal [DT] and transgenic Nirmal [NT]) obtained from the Agrobacterium tumefaciens and Agrobacteriumrhizognenes-mediated transformations, respectively, have been maintained in vitro for 5 years. Plants were studied at regular intervals for various parameters such as plant height, leaf size, multiplication rate, alkaloid profile and presence of marker genes. DT plant gradually lost the GUS gene expression and it was not detected in the fifth year while NT plant demonstrated the presence of genes rolA, rolB and rolC even in the fifth year, indicating the more stable nature of Ri transgene. Vindoline content in the DT was two times more than in non-transformed control plants. Alkaloid and tryptophan profiles were almost constant during the 5 years. The cluster analysis revealed that the DT plant is more close to the control Nirmal plant followed by NT plant. [ABSTRACT FROM AUTHOR]

Tryptophan decarboxylase (TDC) and strictosidine synthase (STR) genes from Catharanthus roseus have been successfully over-expressed in the rol gene integrated cell suspensions of V. minor. Thirty seconds SAAT (sonication-assisted Agrobacterium transformation) treatment of plant cell suspension with LBA1119 having construct () generated three stable TDC + STR over-expressing cell lines-PVG1, PVG2, and PVG3. The transgenes were confirmed by β-glucuronidase GUS histochemical assay and PCR amplification of rol genes/GUS gene. All the three cell suspension lines were found to be slow growing. In comparison to the control cell suspensions (GI = 241.0 ± 5.8), PVG3 cell line registered a growth index (GI) of 208.0 ± 10.0 followed by PVG1 (GI = 140.0 ± 14.2) and PVG2 (GI = 85.0 ± 9.6). The PVG3 cell line was also up-scaled in the 5-l stirred tank bioreactor with GI of 745.6 ± 35.3 under optimized parameters. Only PVG3 line registered a twofold increase in total alkaloid content (2.1 ± 0.1 % dry wt.) and showed vincamine presence (0.003 ± 0.001 % dry wt.) which was further enhanced at the bioreactor level (2.7 ± 0.3 and 0.005 ± 0.001 % dry wt., respectively). Real-time (RT) qPCR analysis of PVG3 showed more than sevenfold to eightfold increase in TDC and STR expression [relative quantity value (RQ) = 7.6 ± 0.8 (TDC); RQ = 8.5 ± 0.9 (STR)]. [ABSTRACT FROM AUTHOR]

• Higher-than-usual vindoline-accumulating genotype of Catharanthus roseus studied. • B present in low amount in the dwarf vindoline-rich plant. • K, Ca, Mg, Fe, Zn, Al and Mn present in higher amount in the vindoline-rich plant. • Vindoline content found to be a direct function of laticifer number. • Study provides a lead towards developing an industrially-useful C. roseus variety. Catharanthus roseus is known for its scarce and expensive anticancer bisindole alkaloids, vincristine and vinblastine, which are derived from the coupling of monomeric terpenoid indole alkaloids, catharanthine and vindoline. Vindoline represents the bottleneck in bisindole semi-synthesis as unlike catharanthine, its biosynthesis is restricted to the plant leaves, which are the only commercially-viable source for it. For reducing the exorbitant cost of bisindoles, identification of C. roseus genotypes accumulating higher-than-usual amount of vindoline is urgently required. Here, one such vindoline-rich genotype (CIMAP866) was identified and characterized. It was comparatively analyzed with elite varieties, Dhawal and Nirmal, for morphology, genetic profile, leaf vindoline content (% dry weight), elemental composition, and expression pattern of alkaloid biosynthetic genes. Its vindoline content (~0.20%) was significantly higher than that of Dhawal (~0.09%) and Nirmal (~0.04%). CIMAP866 possesses dwarf character, spreading/bushy growth, wider canopy, shorter petiole and internodes, more laticifers, higher chlorophyll content, altered content of certain elements and distinct genetic profile, as compared to the other genotypes. Significantly higher dat expression in CIMAP866 was a key determinant for its vindoline-rich nature. Vindoline content was a direct function of dat expression and laticifer number. This study has provided a lead towards developing an industrially-useful C. roseus variety. [Display omitted] [ABSTRACT FROM AUTHOR]

The monoterpene indole alkaloids (MIAs) are generally derived from strictosidine, which is formed by condensation of the terpene moiety secologanin and the indole moiety tryptamine. There are conflicting reports on the limitation of either terpene or indole moiety in the production of MIAs in Catharanthus roseus cell cultures. Formation of geraniol by geraniol synthase (GES) is the first step in secologanin biosynthesis. In this study, feeding of C. roseus leaves with geraniol, but not tryptophan (precursor for tryptamine), increased the accumulation of the MIAs catharanthine and vindoline, indicating the limitation of geraniol in MIA biosynthesis. This was further validated by molecular and in planta characterization of C. roseus GES ( CrGES ). CrGES transcripts exhibited leaf and shoot specific expression and were induced by methyl jasmonate. Virus-induced gene silencing (VIGS) of CrGES significantly reduced the MIA content, which was restored to near-WT levels upon geraniol feeding. Moreover, over-expression of CrGES in C. roseus leaves increased MIA content. Further, CrGES exhibited correlation with MIA levels in leaves of different C. roseus cultivars and has significantly lower expression relative to other pathway genes. These results demonstrated that the transcriptional regulation of CrGES and thus, the in planta geraniol availability plays crucial role in MIA biosynthesis. [ABSTRACT FROM AUTHOR]

• In planta content of alkaloids varies during the winter and summer seasons. • Endophytic consortia improved yields of terpenoid indole alkaloids in periwinkle. • Consortia improved vindoline (winter, summer); vinblastine, vincristine (summer). • Different consortia work differently for cultivars during different seasons. Catharanthus roseus produces medicinally important terpenoid indole alkaloids (TIAs) such as vindoline, vinblastine, and vincristine, but in very small amounts. The various microbial consortium consisting of plant growth-promoting and alkaloid enhancing endophytes were developed in order to enhance the overall productivity of such pharmaceutically important alkaloids. Furthermore, we attempted to identify the endophytic consortia suitable to maximally improve the content and yield of TIAs in field-grown C. roseus cultivars during the winter and summer seasons. The consortium, CATDLF5 + CATPS2 (Curvularia sp. +Bacillus pumilis), improves leaf weight in all C. roseus cultivars during winter and summer. On the alkaloids front, the consortium, CATDLF5 + CATPS2, maximally enhanced the vindoline content and yield in C. roseus cvs. Prabal and Nirmal during both winter and summer seasons; but different consortia were effective for improving vindoline content and yield in cv. Dhawal during different seasons. Whereas co-inoculation of CATDLF6 + CATPR13 (Choanephora infundibulifera + Paenibacillus sp.) and CATDLF5 + CATPS2 consortium maximally enhanced vindoline content and yield, respectively, in C. roseus cv. Dhawal during winter; but during summer, CATDLF6 + V3 (Choanephora infundibulifera + Micrococcus sp.) maximally increased both vindoline content and yield in C. roseus cv. Dhawal. The presence of vincristine and vinblastine are maximally detected in C. roseus plants during summer season. The consortium of CATDLF5 + CATPR13 (Curvularia sp. + Paenibacillus sp.) maximally enhanced the content as well as yield of vincristine and vinblastine in C. roseus cvs. Prabal and Nirmal plants, while CATDLF6 + CATPR13 inoculation showed maximal improvement in the content and yield of vinblastine and vincristine in C. roseus cv. Dhawal plants. This study identifies the best combination of endophytes that can maximize the plant growth and TIAs yield in various C. roseus plant cultivars under field conditions for both winter and summer seasons. [ABSTRACT FROM AUTHOR]