Table 1

The primers information

Fig.1

Expression levels of key genes related to flowering in shoot apical meristems of Pinus tabuliformis at different developmental stages by heatmap

Fig.2

Expression levels of NF-Y family genes in shoot apical meristems of Pinus tabuliformis at different developmental stages by heatmap

Fig.3

Expression patterns and relative transcript levels of PtTFL2，PtCOL1，PtNF-YB8，and PtNF-YC1 genesduring the early stages of flower bud differentiationThe line charts represented transcript abundance（FPKM） obtained from transcriptome sequencing， while bar charts indicated relative expression levels determined by qRT-PCR. All samples were collected from shoot apical meristems at corresponding developmental stages. Each data point represented the mean of three replicates， with error bars indicating standard deviation（SD）.

Table 2

Information of predicted PtNF - YB8 promoter elements

Fig.4

Schematic diagram of the predicted locations of cis-acting elements in the PtNF-YB8 promoterThe colored boxes represented different types of cis-acting regulatory elements. The promoter of PtNF-YB8 gene was depicted by horizontal lines incorporating various colored segments， with each segment signifying a different class of cis-acting elements. A numerical horizontal axis beneath the promoter indicated the distance of these elements relative to the transcription start site （unit： base pairs）， while 5′ and 3′ denoted the orientation of the nucleic acid strand.

Fig.5

Expression level of PtNF-YB8 gene in shoot apical meristems of Pinus tabuliformis after gibberellin treatmentThe line charts represented transcript abundance （FPKM） obtained from transcriptome sequencing， while bar charts indicated relative expression levels determined by qRT-PCR. The ‘CK’ sample consisted of untreated shoot apical meristems collected in mid-October， while the ‘spraying treatment’ sample consisted of shoot apical meristems collected in mid-October， three months after being sprayed with gibberellin4+7. Each data point represented the mean of three replicates， with error bars indicating standard deviation （SD） . *. Significant difference at P<0.05.

Fig.6

Subcellular localization analysis of thePtNF-YB8 proteinThe subcellular localization was observed by transiently expressing the GFP empty vector or the GFP-PtNF-YB8 fusion protein in Nicotiana benthamiana leaf epidermal cells.

Fig.7

Clone and expression analysis of PtNF-YB8 gene in Arabidopsis transgenic linesA. Extraction and integrity analysis of total RNA from Pinus tabuliformis. Distinct 28S and 18S rRNA bands were observed via agarose gel electrophoresis， indicating that the extracted RNA possessed good integrity； B. PCR detection of transgenic Arabidopsis plants. A specific amplicon of approximately 459 bp confirmed the successful integration of PtNF-YB8 gene into the Arabidopsis genome. C. qRT-PCR analysis of PtNF-YB8 expression in confirmed transgenic lines. The expression level of PtNF-YB8 gene was significantly elevated in overexpression plants（OE-PtNF-YB8） compared with the wild type（WT）， with a highly significant difference（P<0.01）. Error bars represented the standard deviation（SD） of three replicates.

Fig.8

Late flowering phenotype of OE-PtNF-YB8 transgenic ArabidopsisPtNF-YB8 overexpression lines（OE-PtNF-YB8，left three pots） and wild-type plants（WT， right three pots） were grown under identical conditions. Photographs show representative plants at 40 d （A） or 60 d （B） since seed imbibition.

Fig.9

Yeast two-hybrid analysis of the interaction between PtNF-YB8 and different PtNF-Y family membersThe AH109 yeast strain was co-transformed with pGADT7-YB8 and various pGBKT7-Y constructs （YC9，YC10，YC4，YA3，YA9）， followed by growth screening on selective media. SD/-Trp/-Leu medium was used to confirm successful co-transformation. SD/-Trp/-Leu/-His/-Ade medium was used to assess protein-protein interactions. Yeast suspensions were spotted at serial dilution factors （100，101，102，103）.