Histochemical, metabolic and ultrastructural changes in leaf patelliform nectaries explain extrafloral nectar synthesis and secretion in Clerodendrum chinense

Abstract Background and Aims Extrafloral nectaries are nectar-secreting structures present on vegetative parts of plants which provide indirect defences against herbivore attack. Extrafloral nectaries in Clerodendrum chinense are patelliform-shaped specialized trichomatous structures. However, a complete understanding of patelliform extrafloral nectaries in general, and of C. chinense in particular, has not yet been established to provide fundamental insight into the cellular physiological machinery involved in nectar biosynthesis and secretory processes. Methods We studied temporal changes in the morphological, anatomical and ultrastructural features in the architectures of extrafloral nectaries. We also compared metabolite profiles of extrafloral nectar, nectary tissue, non-nectary tissue and phloem sap. Further, both in situ histolocalization and normal in vitro activities of enzymes related to sugar metabolism were examined. Key Results Four distinct tissue regions in the nectar gland were revealed from histochemical characterization, among which the middle nectariferous tissue was found to be the metabolically active region, while the intermediate layer was found to be lipid-rich. Ultrastructural study showed the presence of a large number of mitochondria along with starch-bearing chloroplasts in the nectariferous region. However, starch depletion was noted with progressive maturation of nectaries. Metabolite analysis revealed compositional differences among nectar, phloem sap, nectary and non-nectary tissue. Invertase activity was higher in secretory stages and localized in nectariferous tissue and adjacent region. Conclusions Our study suggests extrafloral nectar secretion in C. chinense to be both eccrine and merocrine in nature. A distinct intermediate lipid-rich layer that separates the epidermis from nectary parenchyma was revealed, which possibly acts as a barrier to water flow in nectar. This study also revealed a distinction between nectar and phloem sap, and starch could act as a nectar precursor, as evidenced from enzymatic and ultrastructural studies. Thus, our findings on changing architecture of extrafloral nectaries with temporal secretion revealed a cell physiological process involved in nectar biosynthesis and secretion.