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 (β-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.

Wintersweet (Chimonanthus praecox) is familiar as a garden plant and woody ornamental flower. On account of its unique flowering time and strong fragrance, it has a high ornamental and economic value. Despite a long history of human cultivation, our understanding of wintersweet genetics and molecular biology remains scant, reflecting a lack of basic genomic and transcriptomic data. In this study, we assembled three cDNA libraries, from three successive stages in flower development, designated as the flower bud with displayed petal, open flower and senescing flower stages. Using the Illumina RNA-Seq method, we obtained 21,412,928, 26,950,404, 24,912,954 qualified Illumina reads, respectively, for the three successive stages. The pooled reads from all three libraries were then assembled into 106,995 transcripts, 51,793 of which were annotated in the NCBI non-redundant protein database. Of these annotated sequences, 32,649 and 21,893 transcripts were assigned to gene ontology categories and clusters of orthologous groups, respectively. We could map 15,587 transcripts onto 312 pathways using the Kyoto Encyclopedia of Genes and Genomes pathway database. Based on these transcriptomic data, we obtained a large number of candidate genes that were differentially expressed at the open flower and senescing flower stages. An analysis of differentially expressed genes involved in plant hormone signal transduction pathways indicated that although flower opening and senescence may be independent of the ethylene signaling pathway in wintersweet, salicylic acid may be involved in the regulation of flower senescence. We also succeeded in isolating key genes of floral scent biosynthesis and proposed a biosynthetic pathway for monoterpenes and sesquiterpenes in wintersweet flowers, based on the annotated sequences. This comprehensive transcriptomic analysis presents fundamental information on the genes and pathways which are involved in flower development in wintersweet. And our data provided a useful database for further research of wintersweet and other Calycanthaceae family plants.

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 usedChimonanthus praecoxas 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 ofSHORT VEGETATIVE PHASE(SVP) andFLOWERING 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 inArabidopsiswas used to confirm the function of candidate genes, and early flowering was induced in35S::CpFT1transgenic lines. Our data provide insight into the minimum CR (570 CU) needed for chilling-induced dormancy breaking and its underlying regulatory mechanism inC. praecox, which provides a new tool for the artificial regulation of flowering time and a rich gene resource for controlling chilling-induced blooming.

MADS-box transcriptional regulators play important roles during plant development. Based on phylogenetic reconstruction, the AP1/SEP/AGL6 superclade of floral MADS-box genes underwent one or two duplication events in the common ancestor of the core eudicots. However, the functional evolution of the AP1/SEP/AGL6 superclade in basal eudicots remains uncharacterized. Epimedium sagittatum is a basal eudicot species valued for its medicinal properties and showing unique floral morphology. In this study, structural and functional variation of FUL-like (AP1 subfamily), SEP-like and AGL6-like genes in this species was investigated to further our understanding of flower evolution in angiosperms. Detailed investigations into the microsynteny and evolutionary history of the floral A and E class MADS-box genes in eudicots were undertaken and used to trace their genomic rearrangements. One AP1-like gene, two SEP-like genes and one AGL6-like gene were cloned from E. sagittatum. Their expression patterns were examined using quantitative RT-PCR in different vegetative and reproductive organs at two developmental stages. Yeast two-hybrid assays were carried out among AP1/SEP/AGL6 superclade, AP3/PI and AGAMOUS subfamily members for elucidation of dimerization patterns. In addition, possible formation of a ternary complex involving B class proteins with the A class protein EsFUL-like, the E class SEP-like protein EsAGL2-1 or the AGL6-class protein EsAGL6 were detected using yeast three-hybrid assays. Transgenic Arabidopsis or tobacco plants expressing EsFUL-like, EsAGL2-1 and EsAGL6-like under the cauliflower mosaic virus (CaMV) 35S promoter were generated and analysed. Genomic studies of AP1 syntenic regions in arabidopsis, columbine, strawberry, papaya, peach, grapevine and tomato were conducted for microsyntenic analyses. Sequence and phylogenetic analyses showed that EsFUL-like is a member of the AP1 (A class) subfamily, EsAGL2-1 and EsAGL2-2 belong to the SEP-like (E class) subfamily, and EsAGL6-like belongs to the AGL6 (AGL6 class) subfamily. Quantitative RT-PCR analyses revealed that the transcripts of the four genes are absent, or minimal, in vegetative tissues and are most highly expressed in floral organs. Yeast two-hybrid results revealed that of the eight MADS-box proteins tested, only EsAGL6-like, EsAGL2-1 and EsAGL2 were able to form strong homo- and heterodimers, with EsAGL6-like and EsAGL2-1 showing similar interaction patterns. Yeast three-hybrid analysis revealed that EsFUL1-like, EsAGL6-like and EsAGL2-1 (representing the three major lineages of the Epimedium AGL/SEP/ALG6 superclade) could act as bridging proteins in ternary complexes with both EsAP3-2 (B class) and EsPI (B class), which do not heterodimerize themselves. Syntenic analyses of sequenced basal eudicots, rosids and asterids showed that most AP1-like and SEP-like genes have been tightly associated as neighbours since the origin of basal eudicots. Ectopic expression of EsFUL-like in arabidopsis caused early flowering through endogenous high-level expression of AP1 and formation of secondary flowers between the first and second whorls. Tobacco plants with ectopic expression of EsAGL2-1 showed shortened pistils and styles, as well as axillary and extra petals in the initial flower. This study provides a description of EsFUL-like, EsAGL2-1, EsAGL2-2 and EsAGL6-like function divergence and conservation in comparison with a selection of model core eudicots. The study also highlights how organization in genomic segments containing A and E class genes in sequenced model species has resulted in similar topologies of AP1 and SEP-like gene trees.

Wintersweet (Chimonanthus praecox) is a popular garden plant because of its flowering time, sweet fragrance, and ornamental value. However, research into the molecular mechanism that regulates flower development in wintersweet is still limited. In this study, we sought to investigate the molecular characteristics, expression patterns, and potential functions of two C3H-type zinc finger (CZF) protein genes, CpCZF1 and CpCZF2, which were isolated from the wintersweet flowers based on the flower developmental transcriptome database. CpCZF1 and CpCZF2 were more highly expressed in flower organs than in vegetative tissues, and during the flower development, their expression profiles were associated with flower primordial differentiation, especially that of petal and stamen primordial differentiation. Overexpression of either CpCZF1 or CpCZF2 caused alterations on stamens in transgenic Arabidopsis. The expression levels of the stamen identity-related genes, such as AGAMOUS (AG), PISTILLATA (PI), SEPALLATA1 (SEP1), SEPALLATA2 (SEP2), SEPALLATA3 (SEP3), APETALA1 (AP1), APETALA2 (AP2), and boundary gene RABBIT EAR (RBE) were significantly up-regulated in CpCZF1 overexpression lines. Additionally, the transcripts of AG, PI, APETALA3 SEP1-3, AP1, and RBE were markedly increased in CpCZF2 overexpressed plant inflorescences. Moreover, CpCZF1 and CpCZF2 could interact with each other by using yeast two-hybrid and bimolecular fluorescence complementation assays. Our results suggest that CpCZF1 and CpCZF2 may be involved in the regulation of stamen development and cause the formation of abnormal flowers in transgenic Arabidopsis plants.