Secondary growth is a key characteristic evolved from seed plants and generates secondary xylem—the most abundant tissue on Earth. Recent studies have uncovered xylem developmental lineages in eudicots and magnoliids of angiosperms. However, xylem development in gymnosperms, the other representative clade of seed plants, remained elusive. We performed single-cell transcriptomics for xylem cells of conifers (Cunninghamia lanceolata), the major clade in gymnosperms. Using Seurat and scVI-based cross-species integration, we reconstructed the xylem differentiation trajectories and revealed that the radial system is conserved across seed plants, while the axial system in C. lanceolata exhibits a composite lineage architecture resembling both eudicots and magnoliids. To validate these trajectories, we established a multi-modal spatial framework incorporating spatial transcriptomics, spatial proteomics, and spatial metabolomics. These three spatial layers provided orthogonal evidence confirming cell-type identities and trajectory inference. Additionally, we identified a xylem cell population unique to gymnosperms, suggesting a lineage-specific specialization. Together, our findings uncover a more complex ancestral xylem architecture in gymnosperms and propose a progressive simplification of axial developmental programs from gymnosperms to angiosperms, highlighting a trajectory of reductive evolution in seed plant vascular development.

Chinese fir (Cunninghamia lanceolata (Lamb.) Hook) is one of the main afforestation tree species in southern China. Continuous planting for multiple generations has led to a decrease in the content of available phosphorus in the soil. To adapt to low phosphorus stress, plants develop a series of physiological, biochemical, and developmental responses through self-regulation. Recent studies have shown that miRNAs play a regulatory role in plants’ responses to low phosphorus stress. However, the regulatory mechanism of miRNAs in Chinese fir in response to low phosphorus stress is still unclear. Here, we performed small RNA sequencing on the Chinese fir roots treated with normal phosphorus and low phosphorus and identified a total of 321 miRNAs, including 139 known miRNAs and 182 new miRNAs, with 43 differentially expressed miRNAs (DEMs). Integrative analysis combined with degradome sequencing data revealed that 193 miRNAs (98 known and 95 new) targeted 469 genes, among which 23 DEMs targeted 44 genes. Gene enrichment analysis indicated that under low phosphorus stress, transcription and transcriptional regulation, as well as signal transduction, were significantly activated in Chinese fir. Modules in the miRNA–target pathways, such as miR166/HD-ZIP III, miR169/NFYA7, miR529/SPL, and miR399/UBC23, may be the key regulatory factors in the response to low phosphorus stress in Chinese fir. In addition, we found that PC-3p-1033_8666 was significantly downregulated and that PC-5p-3786_2830 was significantly upregulated, which presumably respond to low phosphorus stress by indirectly affecting phosphorus-related hormone signaling or PSR genes. The identified miRNA–target network and significantly activated pathways in this study provide insights into the post-transcriptional regulatory mechanisms of Chinese fir adapting to low phosphorus environments, which can offer theoretical references for the stress resistance and superior variety breeding of Chinese fir.

Phosphate (Pi) deficiency is one of the most limiting factors for Chinese fir growth and production. Moreover, continuous cultivation of Chinese fir for multiple generations led to the reduction of soil nutrients, which hindered the yield of Chinese fir in southern China. Although NAC (NAM, ATAF, and CUC) transcription factors (TFs) play critical roles in plant development and abiotic stress resistance, it is still unclear how they regulate the response of Chinese fir to phosphate (Pi) starvation. Based on Pi-deficient transcriptome data of Chinses fir root, we identified a NAC transcription factor with increased expression under Pi deficiency, which was obtained by PCR and named ClNAC100. RT-qPCR confirmed that the expression of ClNAC100 in the root of Chinese fir was induced by phosphate deficiency and showed a dynamic change with time. It was positively regulated by ABA and negatively regulated by JA, and ClNAC100 was highly expressed in the roots and leaves of Chinese fir. Transcriptional activation assay confirmed that ClNAC100 was a transcriptional activator. The promoter of ClNAC100 was obtained by genome walking, which was predicted to contain a large number of stress, hormone, and growth-related cis-elements. Tobacco infection was used to verify the activity of the promoter, and the core promoter was located between −1519 bp and −589 bp. We identified 18 proteins bound to the ClNAC100 promoter and 5 ClNAC100 interacting proteins by yeast one-hybrid and yeast two-hybrid, respectively. We speculated that AHL and TIFY family transcription factors, calmodulin, and E3 ubiquitin ligase in these proteins might be important phosphorus-related proteins. These results provide a basis for the further study of the regulatory mechanism and pathways of ClNAC100 under Pi starvation.

Chinese fir (Cunninghamia lanceolata (Lamb.) Hook) is a widely grown gymnosperm in China. Phosphorus (P) is an indispensable nutrient for the growth of Chinese fir. Inorganic phosphate (Pi) deficiency exists in soils of many Chinese fir planting area regions, and the trees themselves have limited efficiency in utilizing P from the soil. Ethylene is important in regulation responses to nutrient deficiencies. However, little is known about how ethylene signals participate in Pi stress in Chinese fir. A total of six different treatments were performed to reveal the transcript levels of Chinese fir under Pi, ethephon (an ethylene-releasing compound), and CoCl₂ (cobalt chloride, an ethylene biosynthesis inhibitor) treatments. We assembled a full-length reference transcriptome containing 22,243 unigenes as a reference for UMI RNA-seq (Digital RNA-seq). There were 586 Differentially Expressed Genes (DEGs) in the Pi starvation (NP) group, while DEGs from additional ethephon or CoCl₂ in NP were 708 and 292, respectively. Among the DEGs in each treatment, there were 83 TFs in these treatment groups. MYB (v-myb avian myeloblastosis viral oncogene homolog) family was the most abundant transcription factors (TFs). Three ERF (Ethylene response factor) family genes were identified when only ethylene content was imposed as a variable. Enrichment analysis indicated that the ascorbate and aldarate metabolism pathway plays a key role in resistance to Pi deficiency. This study provides insights for further elucidating the regulatory mechanism of Pi deficiency in Chinese fir.