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.

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.

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.

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.

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.

Gibberellin Signaling Is Required for Far-Red Light-Induced Shoot Elongation in Pinus tabuliformis Seedlings.

Gibberellin (GA) is known to play an important role in low red/far-red (R:FR) light ratio-mediated hypocotyl and petiole elongation in Arabidopsis (Arabidopsis thaliana). However, the regulatory relationship between low R:FR and GAs remains unclear, especially in gymnosperms. To increase our understanding of the molecular basis of low R:FR-mediated shoot elongation in pines and to determine whether there is an association between low R:FR and GAs action, we explored the morphological and transcriptomic changes triggered by low R:FR, GAs, and paclobutrazol (PAC), a GAs biosynthesis inhibitor, in Pinus tabuliformis seedlings. Transcriptome profiles revealed that low R:FR conditions and GAs have a common set of transcriptional targets in P. tabuliformis We provide evidence that the effect of low R:FR on shoot elongation in P. tabuliformis is at least partially modulated by GAs accumulation, which can be largely attenuated by PAC. GAs are also involved in the cross talk between different phytohormones in the low R:FR response. A GA biosynthesis gene, encoding ent-kaurenoic acid oxidase (KAO), was strongly stimulated by low R:FR without being affected by GAs feedback regulation or the photoperiod. We show that GA signaling is required for low R:FR-induced shoot elongation in P tabuliformis seedlings, and that there are different regulatory targets for low R:FR-mediated GA biosynthesis between conifers and angiosperms.

The transcriptional activity of a temperature-sensitive transcription factor module is associated with pollen shedding time in pine.

It has long been known that the pollen shedding time in pine trees is correlated with temperature, but the molecular basis for this has remained largely unknown. To better understand the mechanisms driving temperature response and to identify the hub regulators of pollen shedding time regulation in Pinus tabuliformis Carr., we identified a set of temperature-sensitive genes by carrying out a comparative transcriptome analysis using six early pollen shedding trees (EPs) and six late pollen shedding trees (LPs) during mid-winter and at three consecutive time points in early spring. We carried out a weighted gene co-expression network analysis and constructed a transcription factor (TF) collaborative network, merging the common but differentially expressed TFs of the EPs and LPs into a joint network. We found five hub genes in the core TF module whose expression was rapidly induced by low temperatures. The transcriptional activity of this TF module was strongly associated with pollen shedding time, and likely to produce the fine balance between cold hardiness and growth activity in early spring. We confirmed the key role of temperature in regulating flowering time and identified a transcription factor module associated with pollen shedding time in P. tabuliformis. This suggests that repression of growth activity by repressors is the main mechanism balancing growth and cold hardiness in pine trees in early spring. Our results provide new insights into the molecular mechanisms regulating seasonal flowering time in pines.