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.

DNA extraction is a fundamental technique in molecular biology. For Chimonanthus praecox—a winter-flowering tree species—extensive and rapid DNA extraction is necessary to support genetic analyses. Currently, DNA extraction in C. praecox primarily relies on the traditional cetyltrimethylammonium bromide (CTAB) method, which is time-consuming and labor-intensive, hindering large-scale DNA extraction work. In this study, the different steps in the CTAB method are compared and evaluated to optimize the C. praecox leaf DNA extraction process. The water bath duration significantly impacts DNA extraction efficiency; the longer the water bath, the higher the DNA concentration. However, a 10-min water bath is sufficient to yield DNA of ideal concentration and purity. Additionally, a single extraction step is appropriate, with a 10-min precipitation at − 20 °C yielding high-quality DNA. Additionally, the pre-treatment step was modified by using a frozen pipette tip to crush samples directly in a centrifuge tube, reducing operational complexity and minimizing liquid nitrogen and sample consumption. Only 25 mg of sample is required, and high-quality DNA from C. praecox leaves can be extracted within 1 h. The amounts of required sample and liquid nitrogen were reduced by 75% and 90%, respectively. Moreover, the time required for the simplified extraction step was reduced by 77.14%. The applicability of the simplified scheme was evaluated using different C. praecox tissues, Calycanthaceae family members, and species of other families. The simplified scheme extracted DNA from the tepals and leaves of C. praecox with higher concentration purity. However, this protocol was biased toward the Chimonanthus family, Nicotiana tabacum, Populus tomentosa, and Lilium brownii. The proposed method enables the rapid and efficient extraction of high-quality DNA from 25 mg of plant leaves and is suitable for multiple species. This method reduces sample and liquid nitrogen consumption, lowering costs while significantly shortening the procedure time and enhancing extraction efficiency. This method is highly suitable for applications involving the extraction of large amounts of low-concentration DNA across various plant species.

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.

Phenylalanine ammonia-lyase (PAL) is the first enzyme involved in the phenylpropanoid pathway and plays important roles in the secondary metabolisms, development and defense of plants. To study the molecular function of PAL in anthocyanin synthesis of Coleus (Solenostemon scutellarioides (L.) Codd), a Coleus PAL gene designated as SsPAL1 was cloned and characterized using a degenerate oligonucleotide primer PCR and RACE method. The full-length SsPAL1 was 2450 bp in size and consisted of one intron and two exons encoding a polypeptide of 711 amino acids. The deduced SsPAL1 protein showed high identities and structural similarities with other functional plant PAL proteins. A series of putative cis-acting elements involved in transcriptional regulation, light and stress responsiveness were found in the upstream regulatory sequence of SsPAL1. Transcription pattern analysis indicated that SsPAL1 was constitutively expressed in all tissues examined and was enhanced by light and different abiotic factors. The recombinant SsPAL1 protein exhibited high PAL activity, at optimal conditions of 60 °C and pH 8.2. Although the levels of total PAL activity and total anthocyanin concentration have a similar variation trend in different Coleus cultivars, there was no significant correlation between them (r = 0.7529, p > 0.1), suggesting that PAL was not the rate-limiting enzyme for the downstream anthocyanin biosynthetic branch in Coleus. This study enables us to further understand the role of SsPAL1 in the phenylpropanoid (flavonoids, anthocyanins) biosynthesis in Coleus at the molecular level.

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.

Salinization, as a serious climate change phenomenon, continues to cause critical issues for soils and crops. Abundance of genetic diversity of plants could contribute in solving the impacts of salinity. However, the relations between physiological and molecular traits of plant species need deep investigation and interpretation. Pot experiments were conducted in the greenhouse to investigate the difference in behavior between two amaranth species when they were beforehand subjected to salinity-stress conditions, specifically on some specific physiological and molecular characteristics. After 45 days from transplanting, salt stress was induced using a 150 mM sodium chloride solution. Treatments were arranged in randomized complete block design using three replications. After 0, 2, 6, 10, and 24 h, samples were collected to evaluate physiological data as well as diversity of gene expression. The samples were immediately frozen in liquid nitrogen and stored in -80⁰ C ultralow temperature refrigerators. Findings revealed that the red amaranth species (Amaranthus cruentus) was more tolerant to saline stress than the green amaranth species (Amaranthus hypochondriacus). Result data revealed that malondialdehyde (MDA) content in the red amaranth increased after 2 h then decreased (after 6 and 10 h) before rising again after 24 h. In the green amaranth, MDA content initially decreased after 2 h before increasing and later decreasing in a zigzag pattern ending in an increase after 24 h. There was an initial steep rise in proline content of green amaranth after 2 h which continued to still rise moderately till 24 h. In the red amaranth the initial rise in proline continued after 2, 6 and 10 h before it stopped. Results also showed that red amaranth gave higher value of superoxide dismutase (SOD) activity as compared with green amaranth in all tested salinity exposure times. The investigation on the expression of four genes assessed through quantitative PCR indicated the efficiency of red amaranth in increasing the expression of SOS1, HKT1, NHX1, and DGR2 genes, which encode adaptation-related proteins under salinity stress. In summary, the current work demonstrated that red amaranth could be an efficient genetic resource in improving salt-tolerant genotypes belonging to Amaranthus genus.