Integrated cytological and transcriptomic analyses reveal the molecular basis of spur variation in Impatiens...

The spur, a key morphological structure in Impatiens uliginosa, plays important functional roles in attracting specific pollinators, enhancing pollination efficiency, and facilitating interspecific reproductive isolation. While its ecological and evolutionary significance has been extensively studied, the cytological and molecular mechanisms underlying spur morphogenesis, particularly in non-model plants, remain poorly understood. In this study, we used wild-type (WT) plants bearing a single spur, two-spur mutant (2SM) and three-spur mutant (3SM) of I. uliginosa. By integrating morphometric analysis, cytological examination, and transcriptome sequencing, we identified candidate genes and hormonal regulatory networks associated with spur variation at the molecular level for the first time. This study provides new insights into the molecular basis of spur formation in I. uliginosa and the genus Impatiens more broadly.

The developmental dynamics of spurs were analyzed both in WT and mutant I. uliginosa, indicating that spur growth follows a typical sigmoidal curve, with lateral spurs being significantly shorter than the main spurs. The cellular development mechanisms were similar between these two spur types: initial spur formation was predominantly driven by cell division, whereas subsequent elongation primarily depended on cell expansion, with the formation of internal cellular protrusions regulated by anisotropic cell growth. Transcriptome sequencing of spurs at the early stage yielded 32.69 Gb of high-quality data, from which 42,721 unigenes were assembled. Functional annotation against the NR, Swiss-Prot, Pfam, COG, GO, and KEGG databases resulted in the annotation of 24,031 genes. Differential expression analysis identified 8,592 differentially expressed genes (DEGs), which were enriched in 371 GO terms and 111 KEGG pathways. Notably, the "plant hormone signal transduction" pathway showed the highest enrichment in the mutant spurs. A total of 955 transcription factors (TFs) belonging to 34 families, including MYB, AP2/ERF, and TCP, were identified. Through qRT-PCR validation, ten candidate genes potentially involved in spur variation were identified, including LTP, GRP5, PDF1, and GID2.

Our study elucidates the morphological and cellular developmental mechanisms of the spurs in WT and mutant I. uliginosa, and identifies a series of candidate genes associated with spur variation, including cell cycle, cell division, cell elongation, and plant hormones. The findings provide valuable data and resources for further unraveling the molecular mechanisms underlying spur variation in Impatiens species.