Volatile benzenoids/phenylpropanoids are the main flower scent compounds in petunia (Petunia hybrida). Heat shock factors (HSFs), well known as the main regulator of heat stress response, have been found to be involved in the biosynthesis of benzenoid/phenylpropanoid and other secondary metabolites. In order to figure out the potential function of HSFs in the regulation of floral scent in petunia, we systematically identified the genome-wide petunia HSF genes and analyzed their expression and then the interaction between the key petunia HSF gene with target gene involved in benzenoid/phenylpropanoid biosynthesis. The results revealed that 34 HSF gene family members were obtained in petunia, and most petunia HSFs contained one intron. The phylogenetic analysis showed that 23 petunia HSFs were grouped into the largest subfamily HSFA, while only two petunia HSFs were in HSFC subfamily. The DBD domain and NLS motif were well conserved in most petunia HSFs. Most petunia HSF genes’ promoters contained STRE motifs, the highest number of cis-acting element. PhHSF19 is highly expressed in petal tubes, followed by peduncles and petal limbs. During flower development, the expression level of PhHSF19 was dramatically higher at earlier flower opening stages than that at the bud stage, suggesting that PhHSF19 may have potential roles in regulating benzenoid/phenylpropanoid biosynthesis. The expression pattern of PhHSF19 is positively related with PhPAL2, which catalyzes the first committed step in the phenylpropanoid pathway. In addition, there are three STRE elements in the promoter of PhPAL2. PhHSF19 was proven to positively regulate the expression of PhPAL2 according to the yeast one hybrid and dual luciferase assays. These results lay a theoretical foundation for further studies of the regulation of HSFs on plant flower scent biosynthesis.

Rhododendron plants have ornamental, commercial, and medicinal value to people. Flavonoids are one of the components used in traditional remedies, and Rhododendron plants are found to be rich in flavonoids. Flavonoids can reduce the risk of human disease and participate in the regulation of antioxidant defense systems in response to heat stress. Rhododendron prefers cold climates, so the relatively high temperatures of cities affect the extraction of medicinal ingredients and limit the cultivation environment. Recent studies found that the exogenous application of calcium acts to alleviate heat stress in Rhododendron plants. This study explores the mechanism by which exogenous calcium alleviates heat stress and the role of flavonoids in regulating the antioxidative system in Rhododendron × pulchrum Sweet using combined transcriptomic and metabolomic methods. The activities of peroxidase, catalase and superoxide enzymes were found to increase in response to heat stress and external CaCl₂ in the leaves of R. × pulchrum. In total, 433 metabolic components and 370 DEGs were identified as being differentially expressed in response to heat stress and external calcium chloride (CaCl₂) in the leaves of R. × pulchrum. These results illustrate that heat stress induces oxidative stress and that external CaCl₂ can enhance the heat tolerance of Rhododendron. Flavonoid compounds are responsible for the antioxidant scavenging of reactive oxygen species in R. × pulchrum leaves exposed to heat stress and external calcium.

(1) Rhododendron is one of the top ten traditional flowers in China, with both high ornamental and economic values. However, with the change of the environment, Rhododendron suffers from various biological stresses. The WRKY transcription factor is a member of the most crucial transcription factor families, which plays an essential regulatory role in a variety of physiological processes and developmental stresses. (2) In this study, 57 RsWRKYs were identified using genome data and found to be randomly distributed on 13 chromosomes. Based on gene structure and phylogenetic relationships, 57 proteins were divided into three groups: I, II, and III. Multiple alignments of RsWRKYs with Arabidopsis thaliana homologous genes revealed that WRKY domains in different groups had different conserved sites. RsWRKYs have a highly conserved domain, WRKYGQK, with three variants, WRKYGKK, WRKYGEK, and WRKYGRK. Furthermore, cis-acting elements analysis revealed that all of the RsWRKYs had stress and plant hormone cis-elements, with figures varying by group. Finally, the expression patterns of nine WRKY genes treated with gibberellin acid (GA), methyl jasmonate (MeJA), heat, and drought in Rhododendron were also measured using quantitative real-time PCR (qRT-PCR). The results showed that the expression levels of the majority of RsWRKY genes changed in response to multiple phytohormones and abiotic stressors. (3) This current study establishes a theoretical basis for future studies on the response of RsWRKY transcription factors to various hormone and abiotic stresses as well as a significant foundation for the breeding of new stress-tolerant Rhododendron varieties.

The heat tolerance of plants can be improved by using exogenous calcium chloride (CaCl₂) to cope with temperature fluctuations. Since global climates continue to warm, it is important to further explore the way in which plants respond to heat stress with the use of CaCl₂. We aimed to explore the effect of exogenous CaCl₂ on the leaf microstructure, leaf epidermal ultrastructure, and chlorophyll a fluorescence of Rhododendron × pulchrum (R. × pulchrum) under heat stress. In the leaves of R. × pulchrum treated with exogenous CaCl₂, compared to the control, the thickness of the epidermis, spongy tissues, and stomatal aperture increased, whereas the stomata density and ratio of closed/open stomata decreased. In the leaves of R. × pulchrum under heat stress conditions, compared to the control, the values of the maximal photochemical efficiency of photosystem II (F_v/F_m), the performance index on an absorption basis (PI_ABS), the quantum yield for the reduction of terminal electron acceptors on the acceptor side of PSI (φ_Ro), and the energy absorbed per unit cross-section of a photosynthesizing object at the moment of achieving the fluorescence maximum (ABS/CS_M) all decreased, whereas the quantum yield of the energy dissipation (φ_Do) increased significantly. However, these differences disappeared when R. × pulchrum was treated with exogenous CaCl₂. This suggests that exogenous CaCl₂ can improve the heat tolerance in R. × pulchrum by regulating the leaf anatomical structure and the behavior of epidermal cells and stomata in leaves, protecting the stability of photosystems I and II and improving the electron transfer from Q_A to Q_B. Our study could provide a theoretical basis for the breeding, further research, and utilization of Rhododendron in the context of global warming.