Micromorphology, histochemistry and ultrastructure of the foliar trichomes of Withania somnifera (L.) Dunal (Solanaceae).

MAIN CONCLUSION: The leaves of Withania somnifera contained four morphologically distinct trichome types: glandular capitate, non-glandular dendritic (branched), non-glandular bicellular and non-glandular multicellular trichomes. Major phytochemical compounds present within glandular and non-glandular trichomes were alkaloids and phenolic compounds. The aim of this study was to characterize the micromorphology of the foliar trichomes of Withania somnifera as well as to elucidate the location and composition of the secretory products. Trichome density and length was also determined in three developmental stages of the leaves. Light microscopy and scanning electron microscopy showed the presence of four morphologically distinct trichome types: glandular capitate, non-glandular dendritic, non-glandular bicellular and non-glandular multicellular. The dendritic trichomes exhibited cuticular warts which are involved in the "Lotus-Effect". Glandular capitate and non-glandular dendritic trichomes were aggregated on the mid-vein of young and mature leaves, possibly to protect underlying vasculature. Histochemical staining also revealed the presence of two major classes of phytochemical compounds that are of medicinal importance, i.e. alkaloids and phenolic compounds. These compounds are used to treat a wide variety of ailments and also act as chemical deterrents in plants. The results of this study explain possible roles of four morphologically distinct trichome types based on their morphology, foliar distribution and content.

Functional evolution of cis-regulatory modules of STMADS11 superclade MADS-box genes.

Evolution of phenotypic morphologies is closely associated with modular organization of cis-regulatory elements underlying expression divergence. The MADS-box gene family is the subject of extensive studies that try to unscramble the structural complexity of flowering plants. This study is envisaged to explore the potential of CRMs in highly constrained non-coding elements of STMADS11superclade MADS-box genes in expression divergence. Phylogenetic reconstruction differentiated the STMADS11 genes into SVP-like, ZMM19-like, MPF1-like and MPF2-like clades. Differential gene expression in vegetative and floral organs was evident within the clades as well as at inter-clade level. The genomic DNA search for clusters of short motifs and sequence conservation of the -2 kb promoter region of particularly, MPF2-like clade permitted to establish three well defined CRMs where transcription factors bind, being CRM1 the activator, CRM2 the repressor, and CRM3 the enhancer element. Similar clusters were also mapped in the large 1st introns in the coding region. Within these CRMs many transcription factor-binding sites, particularly the hotspots for MADS-domain TF binding elements--CArG-boxes, directing sepal specific expression in Arabidopsis--were accrued in the CRM1 of MPF2-like promoters. Site-directed mutagenesis and motif swapping through reporter assays allude towards their implication as functionally active elements. In terms of directional evolution of MPF2-like promoters, CRMs are significantly more conserved than flanking regions, hence, bearing the signatures for purifying selection. Thus, CRMs are the pervasive feature of STMADS11 genes and mutations and/or appearance of new transcription factor binding sites and position of the CRMs are responsible for the divergence in expression patterns in this clade. These results have implications in understanding functional evolution of cis-regulatory modules in plants.

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.

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.