Search

The NAC (NAM, ATAF, and CUC) gene family is one of the largest plant-specific transcription factor families. Its members have various biological functions that play important roles in regulating plant growth and development and in responding to biotic and abiotic stresses. However, their functions in woody plants are not fully understood. In this study, we isolated an NAC family member, the CpNAC1 promoter and gene, from wintersweet. CpNAC1 was localized to the nucleus and showed transcriptional activation activity. qRT-PCR analyses revealed that the gene was expressed in almost all tissues tested, with the highest levels found in mature leaves and flower buds. Moreover, its expression was induced by various abiotic stresses and ABA treatment. Its expression patterns were further confirmed in CpNAC1pro:GUS (β-glucuronidase) plants. Among all the transgenic lines, CpNAC1pro-D2 showed high GUS histochemical staining and activity in different tissues of Arabidopsis. Furthermore, its GUS activity significantly increased in response to various abiotic stresses and ABA treatment. This may be related to the stress-related cis-elements, such as ABRE and MYB, which clustered in the CpNAC1pro-D2 segment, suggesting that CpNAC1pro-D2 is the core segment that responds to abiotic stresses and ABA. In addition, CpNAC1-overexpressed Arabidopsis plants had weaker osmosis tolerance than the wild-type plants, demonstrating that CpNAC1 may negatively regulate the drought stress response in transgenic Arabidopsis. Our results provide a foundation for further analyses of NAC family genes in wintersweet, and they broaden our knowledge of the roles that NAC family genes may play in woody plants.

WRKY transcription factors play critical roles in the physiological processes of plants. Although the roles of WRKYs have been characterized in some model plants, their roles in woody plants, especially wintersweet (Chimonanthus praecox), are largely unclear. In this study, a wintersweet WRKY gene named CpWRKY75 belonging to group IIc was isolated and its characteristics were identified. CpWRKY75 is a nucleus-localized protein, and exhibited no transcriptional activation activity in yeast. CpWRKY75 was highly expressed in flowers at different bloom stages. Ectopic expression of CpWRKY75 significantly promoted the flowering time of transgenic Arabidopsis (Arabidopsis thaliana), as determined by the rosette leaf number and first flower open time. The expression levels of flowering-related genes were quantified by qRT-PCR, and the results suggested that CpWRKY75 had obvious influence on the expression level of MICRORNA156C (MIR156C), SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3 (SPL3) and SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9 (SPL9), FLOWERING LOCUS T (FT), LEAFY (LFY), SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), APETALA1 (AP1), CAULIFLOWER (CAL), and FRUITFULL (FUL). These results suggest that CpWRKY75 might have a flowering time regulation function, and additionally provide a new gene resource for the genetic engineering of woody flowering plants.

Wintersweet (Chimonanthus praecox) is a well-known traditional fragrant plant and a winter-flowering deciduous shrub that originated in China. The five different developmental stages of wintersweet, namely, flower-bud period (FB), displayed petal stage (DP), open flower stage (OF), later blooming period (LB), and wilting period (WP) were studied using a scanning electron microscope (SEM) to determine the distribution characteristics of aroma-emitting nectaries. Results showed that the floral scent was probably emitted from nectaries distributed on the adaxial side of the innermost and middle petals, but almost none on the abaxial side. The nectaries in different developmental periods on the petals differ in numbers, sizes, and characteristics. Although the distribution of nectaries on different rounds of petals showed a diverse pattern at the same developmental periods, that of the nectaries on the same round of petals showed some of regularity. The nectary is concentrated on the adaxial side of the petals, especially in the region near the axis of the lower part of the petals. Based on transcriptional sequence and phylogenetic analysis, we report one nectary development related gene CpCRC (CRABS CLAW), and the other four YABBY family genes, CpFIL (FILAMENTOUS FLOWER), CpYABBY2, CpYABBY5-1, and CpYABBY5-2 in C. praecox (accession no. MH718960-MH718964). Quantitative RT-PCR (qRT-PCR) results showed that the expression characteristics of these YABBY family genes were similar to those of 11 floral scent genes, namely, CpSAMT, CpDMAPP, CpIPP, CpGPPS1, CpGPPS2, CpGPP, CpLIS, CpMYR1, CpFPPS, CpTER3, and CpTER5. The expression levels of these genes were generally higher in the lower part of the petals than in the upper halves in different rounds of petals, the highest being in the innermost petals, but the lowest in the outer petals. Relative expression level of CpFIL, CpCRC, CpYABBY5-1, and CpLIS in the innermost and middle petals in OF stages is significant higher than that of in outer petals, respectively. SEM and qRT-PCR results in C. praecox showed that floral scent emission is related to the distribution of nectaries.

The WRKY transcription factors are one of the most important plant-specific transcription factors and play vital roles in various biological processes. However, the functions of WRKY genes in wintersweet (Chimonanthus praecox) are still unknown. In this report, a group IIc WRKY gene, CpWRKY71, was isolated from wintersweet. CpWRKY71 was localized to the nucleus and possessed transcriptional activation activity. qRT-PCR (quantitative real-time PCR) analysis showed that CpWRKY71 was expressed in all tissues tested, with higher expression in flowers and senescing leaves. During the flower development, the highest expression was detected in the early-withering stage, an obvious expression of CpWRKY71 was also observed in the flower primordia differentiation and the bloom stage. Meanwhile, the expression of CpWRKY71 was influenced by various abiotic stress and hormone treatments. The expression patterns of the CpWRKY71 gene were further confirmed in CpWRKY71pro:GUS (&beta, glucuronidase) plants. Heterologous overexpression of CpWRKY71 in Arabidopsis caused early flowering. Consistent with the early flowering phenotype, the expression of floral pathway integrators and floral meristem identity (FMI) genes were significantly up-regulated in transgenic plants. In addition, we also observed that the transgenic plants of CpWRKY71 exhibited precocious leaf senescence. In conclusion, our results suggested that CpWRKY71 may be involved in the regulation of flowering and leaf senescence in Arabidopsis. Our study provides a foundation for further characterization of CpWRKY genes function in wintersweet, and also enrich our knowledge of molecular mechanism about flowering and senescence in wintersweet.