"Point by point" source: The Chinese pine plantations in North China by evidence from mtDNA.

The geographical variation and domestication of tree species are an important part of the theory of forest introduction, and the tracing of the germplasm is the theoretical basis for the establishment of high-quality plantations. Chinese pine (Pinus tabuliformis Carr.) is an important native timber tree species widely distributed in northern China, but it is unclear exactly where germplasm of the main Chinese pine plantation populations originated. Here, using two mtDNA markers, we analyzed 796 individuals representing 35 populations (matR marker), and 873 individuals representing 38 populations (nad5-1 marker) of the major natural and artificial populations in northern China, respectively (Shanxi, Hebei and Liaoning provinces). The results confirmed that the core position of natural SX* populations ("*" means natural population) in the Chinese pine populations of northern China, the genetic diversity of HB and LN plantations was higher than that of natural SX* populations, and there was a large difference in genetic background within the groups of SX* and LN, HB showed the opposite. More importantly, we completed the "point by point" tracing of the HB and LN plantings. The results indicated that almost all HB populations originated from SX* (GDS*, ZTS*, GCS*, and THS*), which resulted in homogeneity of the genetic background of HB populations. Most of germplasm of the LN plantations originated from LN* (ZJS* and WF*), and the other part originated from GDS* (SX*), resulting in the large differences in the genetic background within the LN group. Our results provided a reliable theoretical basis for the scientific allocation, management, and utilization of Chinese pine populations in northern China, and for promoting the high-quality establishment of Chinese pine plantations.

PagARGOS promotes low-lignin wood formation in poplar.

Wood formation, which occurs mainly through secondary xylem development, is important not only for supplying raw material for the 'ligno-chemical' industry but also for driving the storage of carbon. However, the complex mechanisms underlying the promotion of xylem formation remain to be elucidated. Here, we found that overexpression of Auxin-Regulated Gene involved in Organ Size (ARGOS) in hybrid poplar 84 K (Populus alba × Populus tremula var. glandulosa) enlarged organ size. In particular, PagARGOS promoted secondary growth of stems with increased xylem formation. To gain further insight into how PagARGOS regulates xylem development, we further carried out yeast two-hybrid screening and identified that the auxin transporter WALLS ARE THIN1 (WAT1) interacts with PagARGOS. Overexpression of PagARGOS up-regulated WAT1, activating a downstream auxin response promoting cambial cell division and xylem differentiation for wood formation. Moreover, overexpressing PagARGOS caused not only higher wood yield but also lower lignin content compared with wild-type controls. PagARGOS is therefore a potential candidate gene for engineering fast-growing and low-lignin trees with improved biomass production.

Epigenetic regulation and epigenetic memory resetting during plant rejuvenation.

Reversal of plant developmental status from the mature to the juvenile phase, thus leading to the restoration of the developmental potential, is referred to as plant rejuvenation. It involves multilayer regulation, including resetting gene expression patterns, chromatin remodeling, and histone modifications, eventually resulting in the restoration of juvenile characteristics. Although plants can be successfully rejuvenated using some forestry practices to restore juvenile morphology, physiology, and reproductive capabilities, studies on the epigenetic mechanisms underlying this process are in the nascent stage. This review provides an overview of the plant rejuvenation process and discusses the key epigenetic mechanisms involved in DNA methylation, histone modification, and chromatin remodeling in the process of rejuvenation, as well as the roles of small RNAs in this process. Additionally, we present new inquiries regarding the epigenetic regulation of plant rejuvenation, aiming to advance our understanding of rejuvenation in sexually and asexually propagated plants. Overall, we highlight the importance of epigenetic mechanisms in the regulation of plant rejuvenation, providing valuable insights into the complexity of this process.

Genome-wide identification of the Pinus tabuliformis CONSTANS-like gene family and their potential roles in reproductive cone development.

The CONSTANS-like (COL) genes, as a core transcription factor in the photoperiod regulation pathway, play a key role in plant reproduction development. However, their molecular characterization has rarely been studied in Pinus tabuliformis. Here, 10 PtCOL genes were identified in the P. tabuliformis genome and multiple sequence alignments have indicated that the PtCOL proteins contained highly conserved B-BOX1 and CCT domains. Sequence similarity analysis showed that PtCOL1 and PtCOL3 had the higher similarity with Norway spruce COLs (PaCOL2 and PaCOL1) and Arabidopsis COLs (AtCOL3, 4 and 5), respectively. Phylogeny and gene structure analyses revealed that PtCOLs were divided into three subgroups, each with identical or similar distributions of exons, introns, and motifs. Moreover, 10 PtCOLs were distributed on 6 chromosomes and PtCOL9 has syntenic gene pairs in both Ginkgo biloba and Sequoiadendron giganteum. Interestingly, in transcriptome profiles, most PtCOLs exhibited a diurnal oscillation pattern under both long (LD) and short (SD) day conditions. Additionally, PtCOLs were highly expressed in needles and female cones, and showed different spatial expression patterns. Among the ten PtCOLs, PtCOL1/3 heterologous overexpression Arabidopsis displayed a delayed-flowering phenotype under SD, indicating that they are likely to play a crucial role in the reproductive development. Additionally, PtCOL1 and PtCOL3 were not only capable of interacting with each other, but they were each capable of interacting with themselves. Furthermore, PtCOL1 and PtCOL3 were also involved in the MADS-box protein-protein interaction (PPI) network in P. tabuliformis cone development. Direct interactions of PtDAL11 with PtCOL1/3 impeded PtCOL1/3 translocation into the nucleus. In summary, this study provided comprehensive understanding for the functions of the PtCOL gene family and revealed their biological roles in the photoperiod-dependent P. tabuliformis cone development.

An ethylene-induced NAC transcription factor acts as a multiple abiotic stress responsor in conifer.

The proper response to various abiotic stresses is essential for plants' survival to overcome their sessile nature, especially for perennial trees with very long-life cycles. However, in conifers, the molecular mechanisms that coordinate multiple abiotic stress responses remain elusive. Here, the transcriptome response to various abiotic stresses like salt, cold, drought, heat shock and osmotic were systematically detected in Pinus tabuliformis (P. tabuliformis) seedlings. We found that four transcription factors were commonly induced by all tested stress treatments, while PtNAC3 and PtZFP30 were highly up-regulated and co-expressed. Unexpectedly, the exogenous hormone treatment assays and the content of the endogenous hormone indicates that the upregulation of PtNAC3 and PtZFP30 are mediated by ethylene. Time-course assay showed that the treatment by ethylene immediate precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), activated the expression of PtNAC3 and PtZFP30 within 8 hours. We further confirm that the PtNAC3 can directly bind to the PtZFP30 promoter region and form a cascade. Overexpression of PtNAC3 enhanced unified abiotic stress tolerance without growth penalty in transgenic Arabidopsis and promoted reproductive success under abiotic stress by shortening the lifespan, suggesting it has great potential as a biological tool applied to plant breeding for abiotic stress tolerance. This study provides novel insights into the hub nodes of the abiotic stresses response network as well as the environmental adaptation mechanism in conifers, and provides a potential biofortification tool to enhance plant unified abiotic stress tolerance.

Genome-wide identification of late embryogenesis abundant protein family and their key regulatory network in Pinus tabuliformis cold acclimation.

Cold acclimation is a crucial biological process that enables conifers to overwinter safely. The late embryogenesis abundant (LEA) protein family plays a pivotal role in enhancing freezing tolerance during this process. Despite its importance, the identification, molecular functions and regulatory networks of the LEA protein family have not been extensively studied in conifers or gymnosperms. Pinus tabuliformis, a conifer with high ecological and economic values and with high-quality genome sequence, is an ideal candidate for such studies. Here, a total of 104 LEA genes were identified from P. tabuliformis, and we renamed them according to their subfamily group: PtLEA1-PtLEA92 (group LEA1-LEA6), PtSMP1-PtSMP6 (group seed maturation protein) and PtDHN1-PtDHN6 (group Dehydrin). While the sequence structure of P. tabuliformis  LEA genes are conserved, their physicochemical properties exhibit unique characteristics within different subfamily groupings. Notably, the abundance of low-temperature responsive elements in PtLEA genes was observed. Using annual rhythm and temperature gradient transcriptome data, PtLEA22 was identified as a key gene that responds to low-temperature induction while conforming to the annual cycle of cold acclimation. Overexpression of PtLEA22 enhanced Arabidopsis freezing tolerance. Furthermore, several transcription factors potentially co-expressed with PtLEA22 were validated using yeast one-hybrid and dual-luciferase assays, revealing that PtDREB1 could directly bind PtLEA22 promoter to positively regulate its expression. These findings reveal the genome-wide characterization of P. tabuliformis  LEA genes and their importance in the cold acclimation, while providing a theoretical basis for studying the molecular mechanisms of cold acclimation in conifers.

The methylation landscape of giga-genome and the epigenetic timer of age in Chinese pine.

Epigenetics has been revealed to play a crucial role in the long-term memory in plants. However, little is known about whether the epigenetic modifications occur with age progressively in conifers. Here, we present the single-base resolution DNA methylation landscapes of the 25-gigabase Chinese pine (Pinus tabuliformis) genome at different ages. The result shows that DNA methylation is closely coupled with the regulation of gene transcription. The age-dependent methylation profile with a linearly increasing trend is the most significant pattern of DMRs between ages. Two segments at the five-prime end of the first ultra-long intron in DAL1, a conservative age biomarker in conifers, shows a gradual decline of CHG methylation as the age increased, which is highly correlated with its expression profile. Similar high correlation is also observed in nine other age marker genes. Our results suggest that DNA methylation serves as an important epigenetic signature of developmental age in conifers.

The JASMONATE ZIM-domain-OPEN STOMATA1 cascade integrates jasmonic acid and abscisic acid signaling to regulate drought tolerance by mediating stomatal closure in poplar.

Drought, which directly affects the yield of crops and trees, is a natural stress with a profound impact on the economy. Improving water use efficiency (WUE) and drought tolerance are relatively effective strategies to alleviate drought stress. OPEN STOMATA1 (OST1), at the core of abscisic acid (ABA) signaling, can improve WUE by regulating stomatal closure and photosynthesis. Methyl jasmonate (MeJA) and ABA crosstalk is considered to be involved in the response to drought stress, but the detailed molecular mechanism is insufficiently known. Here, Populus euphratica, which naturally grows in arid and semiarid regions, was selected as the species for studying MeJA and ABA crosstalk under drought. A yeast two-hybrid assay was performed using PeOST1 as bait and a nucleus-localized factor, JASMONATE ZIM-domain protein 2 (PeJAZ2), was found to participate in MeJA signaling by interacting with PeOST1. Overexpression of PeJAZ2 in poplar notably increased water deficit tolerance and WUE in both severe and mild drought stress by regulating ABA signaling rather than ABA synthesis. Furthermore, a PeJAZ2 overexpression line was shown to have greater ABA-induced stomatal closure and hydrogen peroxide (H2O2) production. Collectively, this evidence establishes a mechanism in which PeJAZ2 acts as a positive regulator in response to drought stress via ABA-induced stomatal closure caused by H2O2 production. Our study presents a new insight into the crosstalk of ABA and jasmonic acid signaling in regulating WUE and drought stress, providing a basis of the drought tolerance mechanism of P. euphratica.

Genome-wide TCP transcription factors analysis provides insight into their new functions in seasonal and diurnal growth rhythm in Pinus tabuliformis.

BACKGROUND: Pinus tabuliformis adapts to cold climate with dry winter in northern China, serving as important commercial tree species. The TEOSINTE BRANCHED 1, CYCLOIDEA, and PROLIFERATING CELL FACTOR family(TCP)transcription factors were found to play a role in the circadian clock system in Arabidopsis. However, the role of TCP transcription factors in P. tabuliformis remains little understood. RESULTS: In the present study, 43 TCP genes were identified from P. tabuliformis genome database. Based on the phylogeny tree and sequence similarity, the 43 TCP genes were classified into four groups. The motif results showed that different subfamilies indeed contained different motifs. Clade II genes contain motif 1, clade I genes contain motif 1, 8, 10 and clade III and IV contain more motifs, which is consistent with our grouping results. The structural analysis of PtTCP genes showed that most PtTCPs lacked introns. The distribution of clade I and clade II on the chromosome is relatively scattered, while clade III and clade IV is relatively concentrated. Co-expression network indicated that PtTCP2, PtTCP12, PtTCP36, PtTCP37, PtTCP38, PtTCP41 and PtTCP43 were co-expressed with clock genes in annual cycle and their annual cycle expression profiles both showed obvious seasonal oscillations. PtTCP2, PtTCP12, PtTCP37, PtTCP38, PtTCP40, PtTCP41, PtTCP42 and PtTCP43 were co-expressed with clock genes in diurnal cycle. Only the expression of PtTCP42 showed diurnal oscillation. CONCLUSIONS: The TCP gene family, especially clade II, may play an important role in the regulation of the season and circadian rhythm of P. tabuliformis. In addition, the low temperature in winter may affect the diurnal oscillations.

Phytohormone profiles and related gene expressions after endodormancy release in developing Pinus tabuliformis male strobili.

Development after endo-dormancy release ensures perennial plants, such as forest trees, proper response to environmental changes and enhances their adaptability. In northern hemisphere, megasporophore and microsporophore of conifers undergo dormancy to complete their development. Here combined with transcriptome data, we used high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (ESI-HPLC-MS/MS) to quantitatively analyse the various hormones (Abscisic Acid (ABA), 3-Indoleacetic acid (IAA), Gibberellins (GAs), Cytokinin (CTK), Jasmonic acid (JA) and Salicylic acid (SA)) of Chinese pine (Pinus tabuliformis Carr.) male strobili after endo-dormancy release. More specifically, we analysed endogenous hormones and their related-genes and verified the important role of ABA in plants growth and development. We observed rapid decrease in ABA content after dormancy release, resulting in reducing the inhibitory effect on male strobili growth. Similarly, rapid drop in ABA/GA ratio was observed and was associated with the start of male strobili growth and development. Combined with transcriptome data, we found that HAB2-SnRK2.10 played a central role in the ABA pathway in the entire network of hormones regulating male strobili development. Due to external environment warming, the differentially expressed HAB2-SnRK gene led to ABA content rapid decline, thus initiating male strobili growth. We constructed a network of hormone-regulated development to understand the interactions between hormones after male strobili dormancy release of male strobili. This study provided essential foundations for studying megasporophore and microsporophore growth mechanism after endo-dormancy and offered new ideas for flower development in gymnosperms and angiosperms.

The Chinese pine genome and methylome unveil key features of conifer evolution.

Conifers dominate the world's forest ecosystems and are the most widely planted tree species. Their giant and complex genomes present great challenges for assembling a complete reference genome for evolutionary and genomic studies. We present a 25.4-Gb chromosome-level assembly of Chinese pine (Pinus tabuliformis) and revealed that its genome size is mostly attributable to huge intergenic regions and long introns with high transposable element (TE) content. Large genes with long introns exhibited higher expressions levels. Despite a lack of recent whole-genome duplication, 91.2% of genes were duplicated through dispersed duplication, and expanded gene families are mainly related to stress responses, which may underpin conifers' adaptation, particularly in cold and/or arid conditions. The reproductive regulation network is distinct compared with angiosperms. Slow removal of TEs with high-level methylation may have contributed to genomic expansion. This study provides insights into conifer evolution and resources for advancing research on conifer adaptation and development.

Application of titanium regulates the functional components of photosynthetic apparatus in grafted seedlings of Carya cathayensis Sarg. under shade.

Light acts as a key environmental factor for normal growth and development of plants. Carya cathayensis Sarg. (hickory) faces low light conditions, especially those caused by cloudy or rainy days during the rapid growth period, which has caused adverse effects on its growth. In the current investigation, to alleviate the adverse effects of insufficient light on the cultivation of hickory, anti-hydrolyze stabilized ionic titanium (ASIT) was sprayed on the leaves of the three kinds of grafted seedlings and the non-grafted seedlings of hickory grown under different shade conditions. Results showed that the leaf mass per area and chlorophyll content of grafted hickory seedlings were increased after ASIT application. Rubisco content and photosynthetic rate (Pn) of seedlings grown under shading conditions were positively affected by ASIT treatment, especially on the 45th day of treatment, while the interaction effects of the two parameters between ASIT application and different shade treatments were significant. Titanium accumulation was the highest in roots, followed by leaves, and then in stems, while ASIT had the most significant effects on roots and leaves under 50 ± 5% shade. Severe shading inhibited growth and lead to serious destruction of chloroplast ultrastructure. In addition, the role of ASIT was rootstock-dependent, since ASIT had the weakest mitigation effect on the C/H grafted seedlings. To sum up, the application of ASIT to the grafted seedlings of hickory could improve its ability to resist shade stress.

MADS-box transcription factors MADS11 and DAL1 interact to mediate the vegetative-to-reproductive transition in pine.

The reproductive transition is an important event that is crucial for plant survival and reproduction. Relative to the thorough understanding of the vegetative phase transition in angiosperms, a little is known about this process in perennial conifers. To gain insight into the molecular basis of the regulatory mechanism in conifers, we used temporal dynamic transcriptome analysis with samples from seven different ages of Pinus tabuliformis to identify a gene module substantially associated with aging. The results first demonstrated that the phase change in P. tabuliformis occurred as an unexpectedly rapid transition rather than a slow, gradual progression. The age-related gene module contains 33 transcription factors and was enriched in genes that belong to the MADS (MCMl, AGAMOUS, DEFICIENS, SRF)-box family, including six SOC1-like genes and DAL1 and DAL10. Expression analysis in P. tabuliformis and a late-cone-setting P. bungeana mutant showed a tight association between PtMADS11 and reproductive competence. We then confirmed that MADS11 and DAL1 coordinate the aging pathway through physical interaction. Overexpression of PtMADS11 and PtDAL1 partially rescued the flowering of 35S::miR156A and spl1,2,3,4,5,6 mutants in Arabidopsis (Arabidopsis thaliana), but only PtMADS11 could rescue the flowering of the ft-10 mutant, suggesting PtMADS11 and PtDAL1 play different roles in flowering regulatory networks in Arabidopsis. The PtMADS11 could not alter the flowering phenotype of soc1-1-2, indicating it may function differently from AtSOC1 in Arabidopsis. In this study, we identified the MADS11 gene in pine as a regulatory mediator of the juvenile-to-adult transition with functions differentiated from the angiosperm SOC1.

The Transcriptional Landscape and Hub Genes Associated with Physiological Responses to Drought Stress in Pinus tabuliformis.

Drought stress has an extensive impact on regulating various physiological, metabolic, and molecular responses. In the present study, the Pinus tabuliformis transcriptome was studied to evaluate the drought-responsive genes using RNA- Sequencing approache. The results depicted that photosynthetic rate and H2O conductance started to decline under drought but recovered 24 h after re-watering; however, the intercellular CO2 concentration (Ci) increased with the onset of drought. We identified 84 drought-responsive transcription factors, 62 protein kinases, 17 transcriptional regulators, and 10 network hub genes. Additionally, we observed the expression patterns of several important gene families, including 2192 genes positively expressed in all 48 samples, and 40 genes were commonly co-expressed in all drought and recovery stages compared with the control samples. The drought-responsive transcriptome was conserved mainly between P. tabuliformis and A. thaliana, as 70% (6163) genes had a homologous in arabidopsis, out of which 52% homologous (3178 genes corresponding to 2086 genes in Arabidopsis) were also drought response genes in arabidopsis. The collaborative network exhibited 10 core hub genes integrating with ABA-dependent and independent pathways closely conserved with the ABA signaling pathway in the transcription factors module. PtNCED3 from the ABA family genes had shown significantly different expression patterns under control, mild, prolonged drought, and recovery stages. We found the expression pattern was considerably increased with the prolonged drought condition. PtNCED3 highly expressed in all drought-tested samples; more interestingly, expression pattern was higher under mild and prolonged drought. PtNCED3 is reported as one of the important regulating enzymes in ABA synthesis. The continuous accumulation of ABA in leaves increased resistance against drought was due to accumulation of PtNCED3 under drought stress in the pine needles.

Leveraging breeding programs and genomic data in Norway spruce (Picea abies L. Karst) for GWAS analysis.

BACKGROUND: Genome-wide association studies (GWAS) identify loci underlying the variation of complex traits. One of the main limitations of GWAS is the availability of reliable phenotypic data, particularly for long-lived tree species. Although an extensive amount of phenotypic data already exists in breeding programs, accounting for its high heterogeneity is a great challenge. We combine spatial and factor-analytics analyses to standardize the heterogeneous data from 120 field experiments of 483,424 progenies of Norway spruce to implement the largest reported GWAS for trees using 134 605 SNPs from exome sequencing of 5056 parental trees. RESULTS: We identify 55 novel quantitative trait loci (QTLs) that are associated with phenotypic variation. The largest number of QTLs is associated with the budburst stage, followed by diameter at breast height, wood quality, and frost damage. Two QTLs with the largest effect have a pleiotropic effect for budburst stage, frost damage, and diameter and are associated with MAP3K genes. Genotype data called from exome capture, recently developed SNP array and gene expression data indirectly support this discovery. CONCLUSION: Several important QTLs associated with growth and frost damage have been verified in several southern and northern progeny plantations, indicating that these loci can be used in QTL-assisted genomic selection. Our study also demonstrates that existing heterogeneous phenotypic data from breeding programs, collected over several decades, is an important source for GWAS and that such integration into GWAS should be a major area of inquiry in the future.

Transcriptome-wide isolation and expression of NF-Y gene family in male cone development and hormonal treatment of Pinus tabuliformis.

It is known that nuclear factor Y (NF-Y) transcription factors play an important role in flowering time regulation and hormone response (ABA, GA) in angiosperms, but, little known in conifers. Moreover, the NF-Y gene family has not been comprehensively reported in conifers. Here, we identified 9 NF-YA, 9 NF-YB and 10 NF-YC genes in Pinus tabuliformis using Arabidopsis NF-Y protein sequences as queries. Additionally, by comparing conserved regions and phylogenetic relationships of the PtNF-Ys, we found that NF-Ys were both conserved and altered during evolution. PtTFL2, PtCO, PtNF-YC1 and PtNF-YC4 were exploited by expression profile in male cone development and correlation analysis. Furthermore, NF-YC1/4 and DPL (DELLA protein of P. tabuliformis) were interacted by yeast two-hybrid and BiFC assays, which suggested that NF-YC1/4 may be involved in gibberellins signaling pathway. Moreover, the multiple types of phytohormones-responsive cis-elements (ABA, JA, IAA, SA) have been found, and gene expression profile analysis showed that many NF-Y genes responded positively to SA and as opposed to IAA and JA, revealing the potential role of NF-Ys in conifers resistance. In summary, this study provided the basis for further investigation of the function of NF-Y genes in conifers.

The Tetracentron genome provides insight into the early evolution of eudicots and the formation of vessel elements.

BACKGROUND: Tetracentron sinense is an endemic and endangered deciduous tree. It belongs to the Trochodendrales, one of four early diverging lineages of eudicots known for having vesselless secondary wood. Sequencing and resequencing of the T. sinense genome will help us understand eudicot evolution, the genetic basis of tracheary element development, and the genetic diversity of this relict species. RESULTS: Here, we report a chromosome-scale assembly of the T. sinense genome. We assemble the 1.07 Gb genome sequence into 24 chromosomes and annotate 32,690 protein-coding genes. Phylogenomic analyses verify that the Trochodendrales and core eudicots are sister lineages and showed that two whole-genome duplications occurred in the Trochodendrales approximately 82 and 59 million years ago. Synteny analyses suggest that the γ event, resulting in paleohexaploidy, may have only happened in core eudicots. Interestingly, we find that vessel elements are present in T. sinense, which has two orthologs of AtVND7, the master regulator of vessel formation. T. sinense also has several key genes regulated by or regulating TsVND7.2 and their regulatory relationship resembles that in Arabidopsis thaliana. Resequencing and population genomics reveals high levels of genetic diversity of T. sinense and identifies four refugia in China. CONCLUSIONS: The T. sinense genome provides a unique reference for inferring the early evolution of eudicots and the mechanisms underlying vessel element formation. Population genomics analysis of T. sinense reveals its genetic diversity and geographic structure with implications for conservation.

Comparative transcriptome analyses reveal two distinct transcriptional modules associated with pollen shedding time in pine.

BACKGROUND: Seasonal flowering time is an ecologically and economically important trait in temperate trees. Previous studies have shown that temperature in many tree species plays a pivotal role in regulating flowering time. However, genetic control of flowering time is not synchronised in different individual trees under comparable temperature conditions, the underlying molecular mechanism is mainly to be investigated. RESULTS: In the present study, we analysed the transcript abundance in male cones and needles from six early pollen-shedding trees (EPs) and six neighbouring late pollen-shedding trees (LPs) in Pinus tabuliformis at three consecutive time points in early spring. We found that the EPs and LPs had distinct preferred transcriptional modules in their male cones and, interestingly, the expression pattern was also consistently maintained in needles even during the winter dormancy period. Additionally, the preferred pattern in EPs was also adopted by other fast-growing tissues, such as elongating new shoots. Enhancement of nucleic acid synthesis and stress resistance pathways under cold conditions can facilitate rapid growth and maintain higher transcriptional activity. CONCLUSIONS: During the cold winter and early spring seasons, the EPs were more sensitive to relatively warmer temperatures and showed higher transcriptomic activity than the LPs, indicating that EPs required less heat accumulation for pollen shedding than LPs. These results provided a transcriptomic-wide understanding of the temporal regulation of pollen shedding in pines.