A light in the shadow: the use of Lucifer Yellow technique to demonstrate nectar reabsorption

Abstract Background Nectar reabsorption is a widely known phenomenon, related to the strategy of resource-recovery and also to maintain the nectar homeostasis at the nectary. The method currently performed to demonstrate nectar being reabsorbed involves the use of radioactive tracers applied to the nectary. Although this method works perfectly, it is complex and requires specific supplies and equipment. Therefore, here we propose an efficient method to obtain a visual demonstration of nectar reabsorption, adapting the use of Lucifer Yellow CH (LYCH), a fluorescent membrane-impermeable dye that can enter the vacuole by endocytosis. Results We applied a LYCH solution to the floral nectary (FN) of Cucurbita pepo L., which is a species known for its ability of nectar reabsorption, and to the extrafloral nectary (EFN) of Passiflora edulis Sims which does not reabsorb the secreted nectar. In all tests performed, we observed that LYCH stained the nectary tissues differentially according to the reabsorption ability of the nectary. The treated FN of C. pepo presented a concentrated fluorescence at the epidermis that decreased at the deeper nectary parenchyma, until reaching the vascular bundles, indicating nectar reabsorption in the flowers of the species. In contrast, treated EFN of P. edulis presented fluorescence only at the cuticle surface, indicating that nectar is not reabsorbed by that particular tissue. Conclusion LYCH is an efficient marker to demonstrate nectar reabsorption.

The authors thank the Pró-Reitoria de Pesquisa of the University of São Paulo for the help with the cost of the publishing. PCG and JMRBVA thank the grant provided by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). The authors thank the staff of Instituto de Botânica for providing assistance with cultivated material of P. edulis and Joel Caitano (Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto) for providing material of C. pepo. PCG also thank Wilton José da Rocha Lima (Central Analítica – Instituto de Química, Universidade de São Paulo) for technical assistance with confocal microscopy. JMRBVA thanks Laura de Castro Lamonica for helping with practical work. FB acknowledges the research productivity grant received from the Brazilian National Council for Scientific and Technological Development (CNPq). We thank the two anonymous referees for their valuable comments to the manuscript and their constructive suggestions.

The authors thank the PróReitoria de Pesquisa of the University of São Paulo for the help with the cost of the publishing. PCG and JMRBVA thank the grant provided by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). The authors thank the staff of Instituto de Botânica for providing assistance with cultivated material of P. edulis and Joel Caitano (Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto) for providing material of C. pepo. PCG also thank Wilton José da Rocha Lima (Central Analítica – Instituto de Química, Universidade de São Paulo) for technical assistance with confocal microscopy. JMRBVA thanks Laura de Castro Lamonica for helping with practical work. FB acknowledges the research productivity grant received from the Brazilian National Council for Scientific and Technological Development (CNPq). We thank the two anonymous referees for their valuable comments to the manuscript and their constructive suggestions.