Scientists decode the Ca²⁺ signaling pathway that reduces stone cells
image: Model of the transcriptional regulatory module PuNAC21–PuDof2.5 regulates Ca2+-mediated lignin biosynthesis in pear. Ca2+ inhibits lignin synthesis and decrease stone cell content in pear fruit development through three pathways: (1) Ca2+-induced PuNAC21 suppressing PuPRX42-like transcription activity to reduce lignin synthesis. (2) Ca2+ weakens the transcription of PuDof2.5 by PuNAC21, thereby inhibiting the expression of PuPRX42-like and PuCCoAOMT1 to reduce lignin synthesis. (3) Ca2+ suppressed the transcription of PuPRX42-like and PuCCoAOMT1 by weakening the interaction between PuNAC21 and PuDof2.5, leading to reduce lignin synthesis.
Credit: Horticulture Research
Stone cells formed through lignin deposition are a major factor affecting pear fruit texture and commercial value. This study uncovers the molecular mechanism by which calcium ions (Ca²⁺) suppress stone cell accumulation. The researchers identified PuNAC21—a transcription factor repressed by Ca²⁺—as a central regulator of lignin biosynthesis. PuNAC21 directly activates lignin-related genes and interacts with PuDof2.5 to promote lignin accumulation. Ca²⁺ treatment weakens this regulatory module, reducing lignin levels and ultimately decreasing stone cell formation. These findings provide a mechanistic foundation for improving pear quality through molecular breeding and calcium-based orchard management.
Stone cells arise from lignified secondary cell walls and cause a gritty texture, reduced sweetness, and poor flavor in many pear cultivars. Traditional Pyrus ussuriensis varieties such as ‘Nanguo’ are particularly susceptible to excessive stone cell formation. Lignin biosynthesis is controlled by complex transcriptional networks and key enzymes, including PRX and CCoAOMT, which catalyze essential steps in phenolic polymerization. Although Ca²⁺ has long been associated with lignin modification, its genetic regulatory mechanisms remained poorly understood. Based on these challenges, there is a need for in-depth research on the Ca²⁺-regulated molecular mechanisms underlying stone cell formation.
Researchers from Shenyang Agricultural University, in collaboration with partners in Xinjiang, published (DOI: 10.1093/hr/uhaf102) new findings on July 1, 2025, in Horticulture Research. The study reveals how Ca²⁺ suppresses lignin biosynthesis during stone cell development in pear fruits. The team identified PuNAC21 as a Ca²⁺-responsive transcription factor and demonstrated that it interacts with PuDof2.5 to regulate lignin-associated genes PuPRX42-like and PuCCoAOMT1. Their findings uncover a previously unknown Ca²⁺-mediated regulatory pathway that contributes to improved pear texture by reducing stone cell formation.
Using RNA-seq analysis, gene expression profiling, transgenic pear callus assays, histological staining, and ChIP-qPCR, the researchers demonstrated that Ca²⁺ plays a key inhibitory role in lignin accumulation. Measurements across fruit development showed that stone cell content and lignin levels peaked around 35 days after full bloom, while endogenous Ca²⁺ levels declined. Exogenous CaCl₂ treatment significantly reduced stone cell and lignin content in multiple P. ussuriensis cultivars, with ‘Nanguo’ pear showing the strongest response.
At the molecular level, PuNAC21 was shown to bind directly to the promoters of lignin biosynthesis genes PuPRX42-like and PuCCoAOMT1. PuNAC21 also activates PuDof2.5, forming the PuNAC21–PuDof2.5 transcriptional module that enhances lignin gene expression. Ca²⁺ treatment suppresses PuNAC21 transcription, reduces PuDof2.5 expression, weakens their interaction, and diminishes their promoter-binding capacity. This downregulation reduces lignin deposition and thus lowers stone cell accumulation. The findings provide a clear mechanistic explanation for the long-standing horticultural observation that calcium application improves pear fruit texture.
The research team emphasizes that identifying the PuNAC21–PuDof2.5 regulatory module marks a significant advance in understanding stone cell formation. They note that Ca²⁺ functions not only as a nutrient but also as a signaling molecule capable of reshaping transcriptional networks controlling lignification. By demonstrating how Ca²⁺ weakens this module’s regulatory activity, the study offers a scientific basis for integrating calcium nutrition with molecular breeding strategies aimed at improving pear quality.
This research provides promising genetic targets for breeding pears with reduced stone cell content through marker-assisted selection or gene editing of PuNAC21, PuDof2.5, or downstream lignin genes. The findings also support refined CaCl₂ application strategies to enhance fruit firmness while minimizing lignin deposition. Beyond pears, the Ca²⁺-mediated mechanism may have relevance for other fruit crops where lignification affects quality. By bridging molecular biology with practical orchard management, this study enables the development of high-quality, consumer-preferred fruit varieties.
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References
DOI
Original Source URL
https://doi.org/10.1093/hr/uhaf102
Funding information
This study was supported by the Earmarked Fund for the China Agriculture Research System (CARS-28), National Key Research and Development Program of China (2022YFD1600500), and the Key Technology of Calcium-regulated Quality Development in ‘Nanguoli’ Pear (01042017005).
About Horticulture Research
Horticulture Research is an open access journal of Nanjing Agricultural University and ranked number one in the Horticulture category of the Journal Citation Reports ™ from Clarivate, 2023. The journal is committed to publishing original research articles, reviews, perspectives, comments, correspondence articles and letters to the editor related to all major horticultural plants and disciplines, including biotechnology, breeding, cellular and molecular biology, evolution, genetics, inter-species interactions, physiology, and the origination and domestication of crops.