# Proteomic Analysis of Lotus-Derived NnAP2 Regulation of Soluble Sugar and Starch Content in Potato (Solanum tuberosum)

## TL;DR

This study explores how a transcription factor from lotus plants, called NnAP2, affects starch and sugar levels in potatoes, using proteomic analysis to reveal differences in metabolic pathways.

## Contribution

The study introduces a proteomic framework to understand how allelic variation in NnAP2 influences carbon partitioning and starch accumulation in potato tubers.

## Key findings

- NnAP2CC-OE potato tubers had higher starch and lower soluble sugar levels compared to NnAP2TT-OE.
- Proteomic analysis identified 1715 differentially expressed proteins across tissues, with 88.4% being tissue-specific.
- Key starch-synthesis enzymes were upregulated in NnAP2CC-OE tubers, while soluble sugar pathways were downregulated.

## Abstract

The starch content of lotus (Nelumbo nucifera) rhizomes is a key determinant of their taste and overall quality. In our previous work, a candidate transcription factor, NnAP2, was identified and its coding-region single-nucleotide polymorphisms (SNPs) were significantly associated with rhizome enlargement and carbohydrate-related traits. Owing to limitations in stable genetic transformation systems in lotus, potato (Solanum tuberosum) was employed as a heterologous model to investigate the regulatory role of NnAP2 in starch and soluble sugar metabolism. Overexpression of two allelic variants of the NnAP2 transcription factor (CC and TT) in potato resulted in pronounced differences between CC- and TT-overexpressing lines (NnAP2CC-OE and NnAP2TT-OE) in microtuber carbohydrate composition and proteome dynamics, accompanied by divergence in transgene copy number and substantial variation in transgene expression levels among independent lines. Six months after planting transgenic lines NnAP2CC-OE and NnAP2TT-OE, the NnAP2CC-OE micro-tubers exhibited significantly higher starch content and lower soluble sugar levels compared with NnAP2TT-OE. To uncover the underlying molecular basis, profiling of proteoforms was conducted on leaves, stems and tubers of both genotypes through a label-free proteomic strategy. A total of 51,299 peptides matched to 7292 proteins. Principal component analysis demonstrated clear separation of treatment groups, indicating robust differential accumulation of proteoforms. In total, 1715 differentially expressed proteins (DEPs) were identified across tissues (fold change ≥ 1.5 or ≤0.67, p  <  0.05), of which 1516 (88.4%) were tissue-specific. GO and KEGG enrichment analyses revealed that in leaves, DEPs were enriched for amino sugar metabolism, protein transporter activity and cell-wall macromolecule modification; in stems, enrichment included response to biotic stimulus, defense response and transporter activity; in tubers, DEPs were strongly enriched for carbohydrate metabolic processes, starch and sucrose metabolism, the TCA cycle and nucleotide sugar biosynthesis. Key starch-biosynthetic enzymes (e.g., ADP-glucose pyrophosphorylase, UDP-glucose-4-epimerase) were up-regulated in NnAP2CC-OE tubers relative to NnAP2TT-OE, while soluble sugar synthesis pathways (e.g., trehalose-6-phosphate synthase) were down-regulated. Together, these data suggest that elevated NnAP2CC expression in transgenic potato is associated with allele-dependent shifts in central carbon allocation between starch and soluble sugar pathways, as revealed by comparative analyses between NnAP2CC-OE and NnAP2TT-OE. This study provides a comprehensive proteoform framework for allelic variation in an AP2 transcription factor involved in source–sink carbon partitioning and tuber starch accumulation in potato.