Morphological, cytological, physiological, and molecular evidence to explain how underground fleshy roots expand in herbaceous peony (Paeonia lactiflora Pall.)

The fleshy roots of herbaceous peonies serve as vital storage organs and are the primary source material for Chinese herbal medicine. Moreover, in certain cultivars, these roots can be propagated by root cuttings. However, the specific mechanisms governing the expansion and development of fleshy roots in herbaceous peonies currently remain unclear. To elucidate patterns of changes during the enlargement and development of herbaceous peony fleshy roots, this study assessed fleshy roots in six growth and development periods (P1–P6). By integrating environmental monitoring data, morphological, cytological, physiological and biochemical, as well as molecular characteristics across these developmental periods were compared. Both precipitation and temperature influenced the development of fleshy roots. The fastest enlargement of fleshy roots occurred under the following growth conditions: daily average precipitation = 5.6 mm; monthly average air temperature = 25.6°C; monthly average soil temperature = 26.3°C. Phloem and xylem, produced by the activity of vascular cambium, are important components of fleshy roots. Secondary xylem thickness consistently increased across the six developmental periods, resulting in the radial expansion of fleshy roots. When average monthly air and soil temperatures were about 26°C, starch accumulated gradually in fleshy roots, and carbohydrates were mostly starch-based. However, when the average monthly air and soil temperatures decreased to about 11.0°C, soluble sugar content increased. Endogenous hormones such as IAA, ZR, GA3, MeJA, and ABA played regulatory roles throughout the development of fleshy roots, and their levels and dynamic equilibrium were correlated with expansion rate. Elevated IAA and ZR content, and stabilized IAA/ABA and MeJA/ABA ratios, favored fleshy root expansion. Trends in the expression of three genes that play key roles in the expansion of fleshy roots (PlEXLB3, PlAUX1 and PlWRKY13) were consistent with morphological and physiological data, but PlHXK1 and PlIPT3 also play important roles. These findings lay a foundation for studying, in greater depth, the regulatory mechanisms governing the enlargement of herbaceous peony fleshy roots, and offer practical advice pertaining to field cultivation and management practices of this ornamental plant.