Exogenous calcium alleviates heat stress in carnation via integrated physiological, anatomical and multi-omics modulations

Calcium (Ca 2 + ) is crucial for plant growth and helps regulate response to environmental stress. Heat stress (HS) adversely affects carnation ( Dianthus caryophyllus L.), prompting this study to explore the potential of Ca 2+ to alleviate HS. Initially, carnation seedlings were sprayed with varying concentrations of CaCl 2 , with 15 mM proving to be the most effective treatment for promoting growth and physiological resilience to HS. Further analysis demonstrated that prolonged HS worsened seedlings damage, while Ca 2+ ameliorated this damage. Under 12 d HS, carnation exhibited 48.35 %, 32.67 %, and 6.59 % reductions in aerial part fresh weight, dry weight, and relative water content, respectively. Notably, Ca 2+ pretreatment restored these parameters by 23.96 %, 4.01 %, and 5.88 %. Ca 2+ promoted stomatal opening and preserved chloroplast ultrastructure. HS caused reductions in stomatal length, width, aperture, and area by 33.36 %, 52.11 %, 28.29 %, and 37.53 %, respectively. In contrast, Ca 2+ pretreatment improved these parameters by 33.41 %, 56.60 %, 17.55 %, and 40.88 % compared to the HS group. Transcriptomic analysis revealed Ca²⁺-mediated upregulation of photosynthesis-related pathways, including PSII proteins (Psb) and light-harvesting chlorophyll protein complex (LHC) assembly. Integrated multi-omics indicated Ca²⁺ activated MAPK signaling (via genes like MPK3, MKK9 and WRKY33 ), coordinated phenylpropanoid biosynthesis (e.g., PAL and CHS enzymes), and flavonoid metabolism (e.g., naringenin and hesperetin accumulation). Additionally, Ca²⁺ modulated hormone signaling crosstalk (ABA, JA, SA) to establish a multi-layered thermotolerance network. In conclusion, this study provides novel insights for using exogenous Ca 2+ to alleviate heat damage in carnation and offers technical support for energy-efficient production of this flower. • The possible mechanism of exogenous calcium enhancing the heat tolerance of carnation seedlings. • Ca 2+ promoted the growth of carnation under HS, enhanced antioxidant capacity to alleviate oxidative damage. • Ca 2+ enhanced the photosynthetic capacity of carnation by promoting stomatal opening and protecting chloroplast structure. • Ca 2+ activated phenylpropanoid biosynthesis and hormone signal transduction pathways to regulate HS response.