Overexpression of CpWRKY75 from Chimonanthus praecox Promotes Flowering Time in Transgenic Arabidopsis

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.

CpBBX19, a B-Box Transcription Factor Gene of Chimonanthus praecox, Improves Salt and Drought Tolerance in Arabidopsis

Zinc-finger proteins are important transcription factors in plants, responding to adversity and regulating the growth and development of plants. However, the roles of the BBX gene family of zinc-finger proteins in wintersweet (Chimonanthus praecox) have yet to be elucidated. In this study, a group IV subfamily BBX gene, CpBBX19, was identified and isolated from wintersweet. Quantitative real-time PCR (qRT-PCR) analyses revealed that CpBBX19 was expressed in all tissues and that expression was highest in cotyledons and inner petals. CpBBX19 was also expressed in all flower development stages, with the highest expression detected in early initiating bloom, followed by late initiating bloom and bloom. In addition, the expression of CpBBX19 was induced by different abiotic stress (cold, heat, NaCl, and drought) and hormone (ABA and MeJA) treatments. Heterologous expression of CpBBX19 in Arabidopsis thaliana (Arabidopsis) enhanced the tolerance of this plant to salt and drought stress as electrolyte leakage and malondialdehyde (MDA) concentrations in transgenic Arabidopsis after stress treatments were significantly lower than those in wild-type (WT) plants. In conclusion, this research demonstrated that CpBBX19 plays a role in the abiotic stress tolerance of wintersweet. These findings lay a foundation for future studies on the BBX gene family of wintersweet and enrich understanding of the molecular mechanism of stress resistance in wintersweet.

A General Model for Describing the Ovate Leaf Shape

Many plant species produce ovate leaves, but there is no general parametric model for describing this shape. Here, we used two empirical nonlinear equations, the beta and Lobry–Rosso–Flandrois (LRF) equations, and their modified forms (referred to as the Mbeta and MLRF equations for convenience), to generate bilaterally symmetrical curves along the x-axis to form ovate leaf shapes. In order to evaluate which of these four equations best describes the ovate leaf shape, we used 14 leaves from 7 Neocinnamomum species (Lauraceae) and 72 leaves from Chimonanthus praecox (Calycanthaceae). Using the AIC and adjusted root mean square error to compare the fitted results, the modified equations fitted the leaf shapes better than the unmodified equations. However, the MLRF equation provided the best overall fit. As the parameters of the MLRF equation represent leaf length, maximum leaf width, and the distance from leaf apex to the point associated with the maximum leaf width along the leaf length axis, these findings are potentially valuable for studying the influence of environmental factors on leaf shape, differences in leaf shape among closely related plant species with ovate leaf shapes, and the extent to which leaves are bilaterally symmetrical. This is the first work in which temperature-dependent developmental equations to describe the ovate leaf shape have been employed, as previous studies lacked similar leaf shape models. In addition, prior work seldom attempted to describe real ovate leaf shapes. Our work bridges the gap between theoretical leaf shape models and empirical leaf shape indices that cannot predict leaf shape profiles.

Analysis of Floral Fragrance Compounds of Chimonanthus praecox with Different Floral Colors in Yunnan, China

In order to better understand the floral fragrance compounds of Chimonanthus praecox belonging to genus Chimonanthus of Chimonanaceae in Yunnan, headspace solid-phase microextraction combined with gas chromatography-mass spectrometry was used to analyze these compounds from four C. praecox plants with different floral colors. Thirty-one types of floral fragrance compounds were identified, among which terpenes, alcohols, esters, phenols, and heterocyclic compounds were the main compounds. Interestingly, the floral fragrance compounds identified in the flowers of C. praecox var. concolor included benzyl acetate, α-ocimene, eugenol, indole, and benzyl alcohol. By contrast, the floral fragrance compounds β-ocimene, α-ocimene, and trans-β-ocimene were detected in C. praecox var. patens. Cluster analysis showed that C. praecox var. concolor H1, H2, and C. praecox var. patens H4 were clustered in one group, but C. praecox var. patens H3 was individually clustered in the other group. Additionally, principal component analysis showed that α-ocimene, benzyl alcohol, benzyl acetate, cinnamyl acetate, eugenol, and indole were the main floral fragrance compounds that could distinguish the four C. praecox with different floral colors in Yunnan. This study provides a theoretical basis for further elucidating the mechanism and pathway of the floral fragrance release of C. praecox.

Carotenoid Cleavage Dioxygenase Genes of Chimonanthus praecox, CpCCD7 and CpCCD8, Regulate Shoot Branching in Arabidopsis

Strigolactones (SLs) regulate plant shoot development by inhibiting axillary bud growth and branching. However, the role of SLs in wintersweet (Chimonanthus praecox) shoot branching remains unknown. Here, we identified and isolated two wintersweet genes, CCD7 and CCD8, involved in the SL biosynthetic pathway. Quantitative real-time PCR revealed that CpCCD7 and CpCCD8 were down-regulated in wintersweet during branching. When new shoots were formed, expression levels of CpCCD7 and CpCCD8 were almost the same as the control (un-decapitation). CpCCD7 was expressed in all tissues, with the highest expression in shoot tips and roots, while CpCCD8 showed the highest expression in roots. Both CpCCD7 and CpCCD8 localized to chloroplasts in Arabidopsis. CpCCD7 and CpCCD8 overexpression restored the phenotypes of branching mutant max3-9 and max4-1, respectively. CpCCD7 overexpression reduced the rosette branch number, whereas CpCCD8 overexpression lines showed no phenotypic differences compared with wild-type plants. Additionally, the expression of AtBRC1 was significantly up-regulated in transgenic lines, indicating that two CpCCD genes functioned similarly to the homologous genes of the Arabidopsis. Overall, our study demonstrates that CpCCD7 and CpCCD8 exhibit conserved functions in the CCD pathway, which controls shoot development in wintersweet. This research provides a molecular and theoretical basis for further understanding branch development in wintersweet.

Carotenoid Cleavage Dioxygenase Genes of Chimonanthus praecox, CpCCD7 and CpCCD8, Regulate Shoot Branching in Arabidopsis

Strigolactones (SLs) regulate plant shoot development by inhibiting axillary bud growth and branching. However, the role of SLs in wintersweet (Chimonanthus praecox) shoot branching remains unknown. Here, we identified and isolated two wintersweet genes, CCD7 and CCD8, in-volved in the SL biosynthetic pathway. Quantitative real-time PCR revealed that CpCCD7 and CpCCD8 were down-regulated in wintersweet during branching. When new shoots were formed, expression levels of CpCCD7 and CpCCD8 were almost the same as the control (un-decapitation). CpCCD7 was expressed in all tissues, with the highest expression in shoot tips and roots, while CpCCD8 showed the highest expression in roots. Both CpCCD7 and CpCCD8 localized to chloroplasts in Arabidopsis. CpCCD7 and CpCCD8 overexpression restored the phenotypes of branching mutant max3-9 and max4-1, respectively. CpCCD7 overexpression reduced the rosette branch number, whereas CpCCD8 overexpression lines showed no phenotypic differences compared with wild-type plants. Additionally, the expression of AtBRC1 was significantly up-regulated in transgenic lines, indicating that two CpCCD genes functioned similarly to the homologous genes of the Arabidopsis. Overall, our study demonstrates that CpCCD7 and CpCCD8 exhibit conserved functions in the CCD pathway, which controls shoot development in wintersweet. This research provides a molecular and theoretical basis for further understanding branch development in wintersweet.

Transgene CpNAC68 from Wintersweet (Chimonanthus praecox) Improves Arabidopsis Survival of Multiple Abiotic Stresses

The NAC (NAM, ATAFs, CUC) family of transcription factors (TFs) play a pivotal role in regulating all processes of the growth and development of plants, as well as responses to biotic and abiotic stresses. Yet, the functions of NACs from non-model plant species remains largely uncharacterized. Here, we characterized the stress-responsive effects of a NAC gene isolated from wintersweet, an ornamental woody plant that blooms in winter when temperatures are low. CpNAC68 is clustered in the NAM subfamily. Subcellular localization and transcriptional activity assays demonstrated a nuclear protein that has transcription activator activities. qRT-PCR analyses revealed that CpNAC68 was ubiquitously expressed in old flowers and leaves. Additionally, the expression of CpNAC68 is induced by disparate abiotic stresses and hormone treatments, including drought, heat, cold, salinity, GA, JA, and SA. Ectopic overexpression of CpNAC68 in Arabidopsis thaliana enhanced the tolerance of transgenic plants to cold, heat, salinity, and osmotic stress, yet had no effect on growth and development. The survival rate and chlorophyll amounts following stress treatments were significantly higher than wild type Arabidopsis, and were accompanied by lower electrolyte leakage and malondialdehyde (MDA) amounts. In conclusion, our study demonstrates that CpNAC68 can be used as a tool to enhance plant tolerance to multiple stresses, suggesting a role in abiotic stress tolerance in wintersweet.

Integrated transcriptome and proteome analysis provides insight into chilling-induced dormancy breaking in Chimonanthus praecox

Chilling has a critical role in the growth and development of perennial plants. The chilling requirement (CR) for dormancy breaking largely depends on the species. However, global warming is expected to negatively affect chilling accumulation and dormancy release in a wide range of perennial plants. Here, we used Chimonanthus praecox as a model to investigate the CR for dormancy breaking under natural and artificial conditions. We determined the minimum CR (570 chill units, CU) needed for chilling-induced dormancy breaking and analyzed the transcriptomes and proteomes of flowering and non-flowering flower buds (FBs, anther and ovary differentiation completed) with different CRs. The concentrations of ABA and GA3 in the FBs were also determined using HPLC. The results indicate that chilling induced an upregulation of ABA levels and significant downregulation of SHORT VEGETATIVE PHASE (SVP) and FLOWERING LOCUS T (FT) homologs at the transcript level in FBs when the accumulated CR reached 570 CU (IB570) compared to FBs in November (FB.Nov, CK) and nF16 (non-flowering FBs after treatment at 16 °C for −300 CU), which suggested that dormancy breaking of FBs could be regulated by the ABA-mediated SVP-FT module. Overexpression in Arabidopsis was used to confirm the function of candidate genes, and early flowering was induced in 35S::CpFT1 transgenic lines. Our data provide insight into the minimum CR (570 CU) needed for chilling-induced dormancy breaking and its underlying regulatory mechanism in C. praecox, which provides a new tool for the artificial regulation of flowering time and a rich gene resource for controlling chilling-induced blooming.