Dianthus spiculifolius, a perennial herbaceous flower and a member of the Caryophyllaceae family, has strong resistance to cold and drought stresses. To explore the transcriptional responses of D. spiculifolius to individual and combined stresses, we performed transcriptome sequencing of seedlings under normal conditions or subjected to cold treatment (CT), simulated drought treatment (DT), or their combination (CTDT). After de novo assembly of the obtained reads, 112,015 unigenes were generated. Analysis of differentially expressed genes (DEGs) showed that 2026, 940, and 2346 genes were up-regulated and 1468, 707, and 1759 were down-regulated in CT, DT, and CTDT samples, respectively. Among all the DEGs, 182 up-regulated and 116 down-regulated genes were identified in all the treatment groups. Analysis of metabolic pathways and regulatory networks associated with the DEGs revealed overlaps and cross-talk between cold and drought stress response pathways. The expression profiles of the selected DEGs in CT, DT, and CTDT samples were characterized and confirmed by quantitative RT-PCR. These DEGs and metabolic pathways may play important roles in the response of D. spiculifolius to the combined stress. Functional characterization of these genes and pathways will provide new targets for enhancement of plant stress tolerance through genetic manipulation.

Plant SWEETs (Sugars Will Eventually be Exported Transporters) affect the growth of plants by regulating the transport of sugar from source to sink and its intracellular transport between different organelles. In this study, DsSWEET17 from Dianthus spiculifolius was identified and characterized. Real-time quantitative PCR analysis revealed that the expression of DsSWEET17 was affected by exogenous application of fructose and glucose as well as under salt, osmotic, and oxidation stress. Colocalization experiments showed that the DsSWEET17-GFP (green fluorescent protein) fusion protein was localized to the FM4-64-labeled tonoplasts in Arabidopsis. Compared to the wild type, the transgenic Arabidopsis seedlings overexpressing DsSWEET17 had longer roots, greater fresh weight, and a faster root growth upon exogenous application of fructose. Furthermore, transgenic Arabidopsis seedlings had significantly higher fructose accumulation than was observed for the wild-type seedlings. The analysis of root length revealed that transgenic Arabidopsis had higher tolerance to salt, osmotic, and oxidative stresses. Taken together, our results suggest that DsSWEET17 may be a tonoplast sugar transporter, and its overexpression affects sugar metabolism and confers multiple stress tolerance in Arabidopsis.

Plant SWEETs (sugars will eventually be exported transporters) play a role in plant growth and plant response to biotic and abiotic stresses. In the present study, DsSWEET12 from Dianthus spiculifolius was identified and characterized. Real-time quantitative PCR analysis revealed that DsSWEET12 expression was induced by sucrose starvation, mannitol, and hydrogen peroxide. Colocalization experiment showed that the DsSWEET12-GFP fusion protein was localized to the plasma membrane, which was labeled with FM4-64 dye, in Arabidopsis and suspension cells of D. spiculifolius. Compared to wild type plants, transgenic Arabidopsis seedlings overexpressing DsSWEET12 have longer roots and have a greater fresh weight, which depends on sucrose content. Furthermore, a relative root length analysis showed that transgenic Arabidopsis showed higher tolerance to osmotic and oxidative stresses. Finally, a sugar content analysis showed that the sucrose content in transgenic Arabidopsis was less than that in the wild type, while fructose and glucose contents were higher than those in the wild type. Taken together, our results suggest that DsSWEET12 plays an important role in seedling growth and plant response to osmotic and oxidative stress in Arabidopsis by influencing sugar metabolism.

Low temperature stress adversely affects plant growth and development. Isolation and characterization of cold response genes from cold-tolerant plants help to understand the mechanism underlying low temperature tolerance. In this study, PsCor413pm2, a cold-regulated (COR) gene isolated from Phlox subulata, was transferred to Arabidopsis plants to investigate its function. Real-time quantitative PCR analysis revealed that PsCor413pm2 expression was induced by cold. Subcellular localization revealed that the PsCor413pm2-green fluorescent protein (GFP) fusion protein localized to the plasma membrane in tobacco and Arabidopsis plants. Furthermore, overexpression of PsCor413pm2 in Arabidopsis plants enhanced tolerance to low temperature stress. Transgenic Arabidopsis roots had more influx of Ca2+ after a cold shock than wild-type plants, as shown using non-invasive micro-test technology (NMT). Moreover, the transcription abundance of five COR and two C-repeat (CRT) binding factor (CBF) genes in transgenic Arabidopsis plants was higher than that in the wild-type plants under cold stress. Taken together, our results suggest that overexpression of PsCor413pm2 enhances low temperature tolerance in Arabidopsis plants by affecting Ca2+ flux and the expression of stress-related COR and CBF genes.

Cuticular wax formation on the surface of plant leaves is associated with drought-stress tolerance. The identification of wax biosynthesis-related genes will contribute to the genetic improvement of drought resistance in plants. In this study, we characterize a novel Dianthus spiculifolius mutant with increased cuticular wax. The mutant exhibited stronger drought resistance as indicated by less leaf wilting and death, higher leaf relative water content and water retention capacity, and slower water loss and chlorophyll extraction than did the wild type during drought treatment. In the mutant leaves, 2 730 upregulated and 2 151 downregulated differentially expressed genes (DEGs) were identified by transcriptome sequencing. A wax biosynthesis pathway of the identified DEGs was significantly enriched. Finally, three key genes (DsCER1, DsMAH1, and DsWSD1) involved in wax biosynthesis were identified and verified by qPCR. These results suggest that differential expression of DEGs involved in wax biosynthesis may be associated with the increase in cuticular wax in the mutant. Taken together, our results help elucidate wax formation patterns in D. spiculifolius. Furthermore, the DEGs involved in wax biosynthesis identified here may be valuable genetic resources for improving plant stress tolerance through increased accumulation of cuticular wax.