Background: Starch in the lotus seed contains a high proportion of amylose, which endows lotus seed a promising property in the development of hypoglycemic and low-glycemic index functional food. Currently, improving starch content is one of the major goals for seed-lotus breeding. ADP-glucose pyrophosphorylase (AGPase) plays an essential role in regulating starch biosynthesis in plants, but little is known about its characterization in lotus. Results: We describe the nutritional compositions of lotus seed among 30 varieties with starch as a major component. Comparative transcriptome analysis showed that AGPase genes were differentially expressed in two varieties (CA and JX) with significant different starch content. Seven putative AGPase genes were identified in the lotus genome (Nelumbo nucifera Gaertn.), which could be grouped into two subfamilies. Selective pressure analysis indicated that purifying selection acted as a vital force in the evolution of AGPase genes. Expression analysis revealed that lotus AGPase genes have varying expression patterns, with NnAGPL2a and NnAGPS1a as the most predominantly expressed, especially in seed and rhizome. NnAGPL2a and NnAGPS1a were co-expressed with a number of starch and sucrose metabolism pathway related genes, and their expressions were accompanied by increased AGPase activity and starch content in lotus seed. Conclusions: Seven AGPase genes were characterized in lotus, with NnAGPL2a and NnAGPS1a, as the key genes involved in starch biosynthesis in lotus seed. These results considerably extend our understanding on lotus AGPase genes and provide theoretical basis for breeding new lotus varieties with high-starch content.

WUSCHEL-related homeobox (WOX) genes are a class of plant-specific transcription factors, regulating the development of multiple tissues. However, the genomic characterizations and expression patterns of WOX genes have not been analyzed in lotus. In this study, 15 NnWOX genes were identified based on the well-annotated reference genome of lotus. According to the phylogenetic analysis, the NnWOX genes were clustered into three clades, i.e., ancient clade, intermediate clade, and WUS clade. Except for the conserved homeobox motif, we further found specific motifs of NnWOX genes in different clades and divergence gene structures, suggesting their distinct functions. In addition, two NnWOX genes in the ancient clade have conserved expression patterns and other NnWOX genes exhibit different expression patterns in lotus tissues, suggesting a low level of functional redundancy in lotus WOX genes. Furthermore, we constructed the gene co-expression networks for each NnWOX gene. Based on weighted gene co-expression network analysis (WGCNA), ten NnWOX genes and their co-expressed genes were assigned to the modules that were significantly related to the cotyledon and seed coat. We further performed RT-qPCR experiments, validating the expression levels of ten NnWOX genes in the co-expression networks. Our study reveals comprehensive genomic features of NnWOX genes in lotus, providing a solid basis for further function studies.

The yellow flower lotus (Nelumbo lutea) is the sister species of the sacred lotus (N. nucifera). The evolution of gene expression patterns across multiple tissues during the species divergence of these two lotuses remains unexplored. The WUSCHEL-related homeobox (WOX) family, a plant-specific transcription factor family, plays a crucial role in tissue development and stress responses. In this study, utilizing a chromosome-level reference genome and a transcriptome database covering multiple tissues, we identified and categorized 11 NlWOX genes into three subfamilies. We identified seven syntenic gene pairs between NnWOXs and NlWOXs that originated from whole-genome duplications. Through conserved motif analysis, we found subfamily-specific motifs in the protein sequences of NnWOXs and NlWOXs. Variations in the three-dimensional conformations of homologous WOX genes indicate function divergences between the two lotus species. The gene expression matrix of NlWOX across tissues reveals expression divergences within N. lutea and between the two lotus species. By employing a weight gene co-expression network analysis pipeline, we developed eight NlWOX co-expression networks that differed from the co-expression networks of their syntenic genes. Overall, our findings suggest that genomic variations in the WOX orthologs contribute to the distinct expression patterns and regulatory networks observed during the evolution of these two lotuses.