The lotus (Nelumbo Adans.) is a perennial aquatic plant with important value in horticulture, medicine, food, religion, and culture. It is rich in germplasm and more than 2000 cultivars have been cultivated through hybridization and natural selection. Microsporogenesis and male gametogenesis in the anther are important for hybridization in flowering plants. However, little is known about the cytological events, especially related to the stamen, during the reproduction of the lotus. To better understand the mechanism controlling the male reproductive development of the lotus, we investigated the flower structure of the Asian lotus (N. nucifera). The cytological analysis of anther morphogenesis showed both the common and specialized cytological events as well as the formation of mature pollen grains via meiosis and mitosis during lotus anther development. Intriguingly, an anatomical difference in anther appendage structures was observed between the Asian lotus and the American lotus (N. lutea). To facilitate future study on lotus male reproduction, we categorized pollen development into 11 stages according to the characterized cytological events. This discovery expands our knowledge on the pollen and appendage development of the lotus as well as improving the understanding of the species differentiation of N. nucifera and N. lutea.

The lotus (Nelumbo Adans.) is an important aquatic plant with ornamental, medicinal and edible values and cultural connotations. It has single-, semi-double-, double- and thousand-petalled types of flower shape and is an ideal material for developmental research of flower doubling. The lotus is a basal eudicot species without a morphological difference between the sepals and petals and occupies a critical phylogenetic position in flowering plants. In order to investigate the genetic relationship between the sepals and petals in the lotus, the class E genes which affect sepal formation were focused on and analyzed. Here, SEPALLATA 1(NnSEP1) and its homologous genes AGAMOUS-LIKE MADS-BOXAGL9 (NnAGL9) and MADS-BOX TRANSCRIPTION FACTOR 6-like (NnMADS6-like) of the class E gene family were isolated from the flower buds of the Asian lotus (Nelumbo nucifera Gaertn.). The protein structure, subcellular localization and expression patterns of these three genes were investigated. All three genes were verified to locate in the nucleus and had typical MADS-box characteristics. NnSEP1 and NnMADS6-like were specifically expressed in the sepals, while NnAGL9 was highly expressed in the petals, suggesting that different developmental mechanisms exist in the formation of the sepals and petals in the lotus. The significant functional differences between NnSEP1, NnMADS6-like and NnAGL9 were also confirmed by a yeast two-hybrid assay. These results expand our knowledge on the class E gene family in sepal formation and will benefit fundamental research on the development of floral organs in Nelumbo.

ABC transporters (ATP-binding cassette transporters) constitute one of the largest known protein families and are widely distributed in plants. Their primary function involves utilizing energy derived from ATP hydrolysis to transport substrates across membranes against concentration gradients. These transporters play crucial roles in the translocation and accumulation of metabolites, stress tolerance, disease resistance, and plant defense. Lotus is an important traditional Chinese medicinal herb and contains active ingredients primarily composed of secondary metabolites, whose transport and accumulation require the involvement of ABC transporters. However, the function of these ABC transporters remains unexplored in lotus. In this study, 122 ABC transporter genes were predicted within the lotus genome. We identified 1~15 conserved motifs among the NnABC proteins and most of them were stable proteins predominantly located on the plasma membrane with ExPASy-ProtParam, ProComp and WoLF PSORT analysis. Phylogenetic tree analysis revealed that the lotus ABC transporter gene family could be divided into eight subfamilies, from ABCA to ABCI, and the evolution was predominantly driven by purifying selection. Comparative transcriptome analysis between the cultivar ‘Yindu Zhimi’ with orange-reddish stamen and ‘Weishan Hong’ with yellowish stamen, along with quantitative real-time PCR results, showed that the NnABCG25 gene is highly specifically expressed in the orange-reddish stamen. Molecular docking demonstrated that NnABCG25 has a stable affinity for lycopene, β-carotene and β-apocarotenal, suggesting its potential involvement in the transport of carotenoids in the stamen. These findings expand our understanding of the role of ABC transporters in the transport and accumulation of carotenoids, as well as providing a valuable reference for research on the ABC transporter gene family in other plants.