Virus-Induced Silencing of Key Genes Leads to Differential Impact on Withanolide Biosynthesis in the Medicinal Plant, Withania somnifera.

Withanolides are a collection of naturally occurring, pharmacologically active, secondary metabolites synthesized in the medicinally important plant, Withania somnifera. These bioactive molecules are C28-steroidal lactone triterpenoids and their synthesis is proposed to take place via the mevalonate (MVA) and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways through the sterol pathway using 24-methylene cholesterol as substrate flux. Although the phytochemical profiles as well as pharmaceutical activities of Withania extracts have been well studied, limited genomic information and difficult genetic transformation have been a major bottleneck towards understanding the participation of specific genes in withanolide biosynthesis. In this study, we used the Tobacco rattle virus (TRV)-mediated virus-induced gene silencing (VIGS) approach to study the participation of key genes from MVA, MEP and triterpenoid biosynthesis for their involvement in withanolide biosynthesis. TRV-infected W. somnifera plants displayed unique phenotypic characteristics and differential accumulation of total Chl as well as carotenoid content for each silenced gene suggesting a reduction in overall isoprenoid synthesis. Comprehensive expression analysis of putative genes of withanolide biosynthesis revealed transcriptional modulations conferring the presence of complex regulatory mechanisms leading to withanolide biosynthesis. In addition, silencing of genes exhibited modulated total and specific withanolide accumulation at different levels as compared with control plants. Comparative analysis also suggests a major role for the MVA pathway as compared with the MEP pathway in providing substrate flux for withanolide biosynthesis. These results demonstrate that transcriptional regulation of selected Withania genes of the triterpenoid biosynthetic pathway critically affects withanolide biosynthesis, providing new horizons to explore this process further, in planta.

Elucidation of genotype-environment interactions and genetic stability parameters for yield, quality and agromorphological traits in ashwagandha (Withania somnifera (L.) Dunal).

The present study was undertaken to delineate genotype-environment interactions and stability status of 16 genotypes of ashwagandha (Withania somnifera (L.) Dunal) in context to the 12 characters, namely plant height, number of primary branches, number of secondary branches, days to flowering, days to maturity, number of berries, number of seeds/berry, root length, root diameter, root branches, dry root yield and total alkaloid content (%). Experiment was carried out in a randomized complete block design with three replicationsover three different locations (S. K. Nagar, Jagudan and Bhiloda) in north Gujarat for three years (2016-17, 2017-18 and 2018-19). Pooled analysis of variance revealed that the mean squares due to genotypes and genotype 9 environment interaction along with linear and nonlinear components were highly significant (P<0.01) for most of the traits under study. Stability parameters for component traits through Eberhart and Russell model showed that genotypes that can be used directly in breeding programme are SKA-4 for early flowering, SKA-21 for early maturity and SKA-1, SKA-4, SKA-6 and SKA-17 for shorter plant height. Further, SKA-21 could be used for improving number of primary branches per plant, SKA-11 and SKA-17 for number of secondary branches per plant, SKA-19 for number of berries per plant, SKA-6, SKA-21, SKA-27 and AWS-1 for root branches and SKA-17 for root length as these genotypes were found to be moststable across the environments for mentioned traits. The result revealed that some reliable predictions about genotype 9 environment interaction and its unpredictable components were involved significantly in determining the stability of genotypes. Hence, the present investigation can be exploited for the identification of more productive genotypes in specific environments, leading to significant increase in root productivity of ashwagandha.

Plant hormones including ethylene are recruited in calyx inflation in Solanaceous plants.

Plant hormones direct many processes of floral and post-floral morphogenesis in Angiosperms. However, their role in shaping floral morphological novelties, such as inflated calyx syndrome (ICS) exhibited by a few genera of the Solanaceae, remains unknown. In Withania and Physalis, sepals resume growth after pollination and encapsulate the mature fruit to form a balloon-like structure, i.e. ICS. The epidermal cells of calyx show enlargement and lobation post-fertilization. Application of hormones to depistillated flower buds of Withania revealed that cytokinins and gibberellins mimic fertilization signals. The ICS development is a synchronous step with fruit development; both processes are under the control of more or less the same set of hormones, including cytokinins and gibberellic acids. Interestingly, inhibition of ethylene in the system is sufficient to yield inflated calyx in Withania. In contrast, Tubocapsicum, a closely related species and an evolutionary natural loss mutant of ICS - showed no response to applied hormones, and ethylene led to inflation of the receptacle indirectly. In addition to hormones, the expression of an MPF2-like MADS-box transcription factor in sepals is essential for ICS formation. Nevertheless, the interactions between MPF2-like genes and hormones are barely detectable at the transcript level. Our data provide insight into the role of hormones in generating floral morphological diversity during evolution.

Changes in morphological phenotypes and withanolide composition of Ri-transformed roots of Withania somnifera.

Developmental variability was introduced into Withania somnifera using genetic transformation by Agrobacterium rhizogenes, with the aim of changing withasteroid production. Inoculation of W. somnifera with A. rhizogenes strains LBA 9402 and A4 produced typical transformed root lines, transformed callus lines, and rooty callus lines with simultaneous root dedifferentiation and redifferentiation. These morphologically distinct transformed lines varied in T-DNA content, growth rates, and withasteroid accumulation. All of the lines with the typical transformed root morphology contained the T(L) T-DNA, and 90% of them carried the T(R) T-DNA, irrespective of the strain used for infection. Accumulation of withaferin A was maximum (0.44% dry weight) in the transformed root line WSKHRL-1. This is the first detection of withaferin A in the roots of W. somnifera. All of the rooty callus lines induced by strain A4 contained both the T(L) and the T(R)-DNAs. In contrast, 50% of the rooty-callus lines obtained with strain LBA 9402 contained only the T(R) T-DNA. All the rooty callus lines accumulated both withaferin A and withanolide D. The callusing lines induced by LBA 9402 lacked the T(L) T-DNA genes, while all the callusing lines induced by strain A4 contained the T(L) DNA. Four of these callus lines produced both withaferin A (0.15-0.21% dry weight) and withanolide D (0.08-0.11% dry weight), and they grew faster than the transformed root lines. This is the first report of the presence of withasteroids in undifferentiated callus cultures of W. somnifera.

Evolution of the inflated calyx syndrome in Solanaceae.

Species that express the inflated calyx syndrome (ICS) are found in several genera of the Solanaceae. The MADS-box protein MPF2, together with the plant hormones cytokinin and gibberellin, has been shown to be responsible for this trait in Physalis floridana. We have used sequence data from 114 species belonging to 35 genera to construct a molecular phylogeny of Solanaceae. Apart from the 2 Witheringia species analyzed, species within a given genus cluster together on the resulting cladogram. Witheringia solanacea is embedded within the Physalinae, but Witheringia coccoloboides is placed basal to the Iochrominae. The ICS trait seems to be of multiple origins both within the Solanaceae and the Physaleae. Surprisingly, expression of MPF2-like genes in floral organs appears to be plesiomorphic in both the Physaleae and the Capsiceae. Some species in these tribes that show neither ICS nor calyx accrescence fail to express the MPF2-like gene in floral organs. Among those that do express this gene in the calyx are the species Capsicum baccatum, Lycianthes biflora, Tubocapsicum anomalum, W. solanacea, and Vassobia breviflora, all of which form small calyces that do not respond to externally applied hormones. The plesiomorphic nature of MPF2-like gene expression in the calyx of the Physaleae and Capsiceae raises the possibility that originally ICS also was actually a plesiomorphic character in these 2 groups. However, this trait might have undergone changes in a number of species due to secondary loss of components in ICS formation, like hormone response of calyx development. These findings are discussed in an evolutionary context of a molecular pathway leading to ICS.