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

Extrafloral nectaries are nectar-secreting structures present on vegetative parts of the plants which provide indirect defences against herbivore attack. Extrafloral nectaries in Clerodendrum chinense are patelliform-shaped specialised trichomatous structures. However, complete portrayal of patelliform extrafloral nectaries in general and C. chinense in particular, is not yet established for gaining a fundamental insight into the cellular physiological machinery involved in nectar biosynthesis and secretory process.We studied temporal changes in morphological, anatomical and ultrastructural features in 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.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 nectariferous region. However, starch depletion was noticed with the progressing 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.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 distinction between nectar and phloem sap, and starch could act as nectar precursor as evidenced from enzymatic and ultrastructural studies. Thus, our findings on changing architecture with temporal secretion of extrafloral nectaries revealed a cell physiological process involved in nectar biosynthesis and secretion.