The super-pangenome of Populus unveils genomic facets for its adaptation and diversification in widespread forest trees.

Understanding the underlying mechanisms and links between genome evolution and adaptive innovations stands as a key goal in evolutionary studies. Poplars, among the world's most widely distributed and cultivated trees, exhibit extensive phenotypic diversity and environmental adaptability. In this study, we present a genus-level super-pangenome comprising 19 Populus genomes, revealing the likely pivotal role of private genes in facilitating local environmental and climate adaptation. Through the integration of pangenomes with transcriptomes, methylomes, and chromatin accessibility mapping, we unveil that the evolutionary trajectories of pangenes and duplicated genes are closely linked to local genomic landscapes of regulatory and epigenetic architectures, notably CG methylation in gene-body regions. Further comparative genomic analyses have enabled the identification of 142 202 structural variants across species that intersect with a significant number of genes and contribute substantially to both phenotypic and adaptive divergence. We have experimentally validated a ∼180-bp presence/absence variant affecting the expression of the CUC2 gene, crucial for leaf serration formation. Finally, we developed a user-friendly web-based tool encompassing the multi-omics resources associated with the Populus super-pangenome (http://www.populus-superpangenome.com). Together, the present pioneering super-pangenome resource in forest trees not only aids in the advancement of breeding efforts of this globally important tree genus but also offers valuable insights into potential avenues for comprehending tree biology.

Chromatin accessibility dynamics insight into crosstalk between regulatory landscapes in poplar responses to multiple treatments.

Perennial trees develop and coordinate endogenous response signaling pathways, including their crosstalk and convergence, to cope with various environmental stresses which occur simultaneously in most cases. These processes are involved in gene transcriptional regulations that depend on dynamic interactions between regulatory proteins and corresponding chromatin regions, but the mechanisms remain poorly understood in trees. In this study, we detected chromatin regulatory landscapes of poplar under abscisic acid, methyl jasmonate, salicylic acid and sodium chloride (NaCl) treatment, through integrating ATAC-seq and RNA-seq data. Our results showed that the degree of chromatin accessibility for a given gene is closely related to its expression level. However, unlike the gene expression that shows treatment-specific response patterns, changes in chromatin accessibility exhibit high similarities under these treatments. We further proposed and experimentally validated that a homologous gene copy of RESPONSIVE TO DESICCATION 26 mediates the crosstalk between jasmonic acid and NaCl signaling pathways by directly regulating the stress-responsive genes and that circadian clock-related transcription factors like REVEILLE8 play a central role in response of poplar to these treatments. Overall, our study provides a chromatin insight into the molecular mechanism of transcription regulatory networks in response to different environmental stresses and raises the key roles of the circadian clock of poplar to adapt to adverse environments.

The C2 H2 -type zinc finger transcription factor OSIC1 positively regulates stomatal closure under osmotic stress in poplar.

Salt and drought impair plant osmotic homeostasis and greatly limit plant growth and development. Plants decrease stomatal aperture to reduce water loss and maintain osmotic homeostasis, leading to improved stress tolerance. Herein, we identified the C2 H2 transcription factor gene OSMOTIC STRESS INDUCED C2 H2 1 (OSIC1) from Populus alba var. pyramidalis to be induced by salt, drought, polyethylene glycol 6000 (PEG6000) and abscisic acid (ABA). Overexpression of OSIC1 conferred transgenic poplar more tolerance to high salinity, drought and PEG6000 treatment by reducing stomatal aperture, while its mutant generated by the CRISPR/Cas9 system showed the opposite phenotype. Furthermore, OSIC1 directly up-regulates PalCuAOζ in vitro and in vivo, encoding a copper-containing polyamine oxidase, to enhance H2 O2 accumulation in guard cells and thus modulates stomatal closure when stresses occur. Additionally, ABA-, drought- and salt-induced PalMPK3 phosphorylates OSIC1 to increase its transcriptional activity to PalCuAOζ. This regulation of OSIC1 at the transcriptional and protein levels guarantees rapid stomatal closure when poplar responds to osmotic stress. Our results revealed a novel transcriptional regulatory mechanism of H2 O2 production in guard cells mediated by the OSIC1-PalCuAOζ module. These findings deepen our understanding of how perennial woody plants, like poplar, respond to osmotic stress caused by salt and drought and provide potential targets for breeding.

Genomic insights into local adaptation and future climate-induced vulnerability of a keystone forest tree in East Asia.

Rapid global climate change is posing a substantial threat to biodiversity. The assessment of population vulnerability and adaptive capacity under climate change is crucial for informing conservation and mitigation strategies. Here we generate a chromosome-scale genome assembly and re-sequence genomes of 230 individuals collected from 24 populations for Populus koreana, a pioneer and keystone tree species in temperate forests of East Asia. We integrate population genomics and environmental variables to reveal a set of climate-associated single-nucleotide polymorphisms, insertion/deletions and structural variations, especially numerous adaptive non-coding variants distributed across the genome. We incorporate these variants into an environmental modeling scheme to predict a highly spatiotemporal shift of this species in response to future climate change. We further identify the most vulnerable populations that need conservation priority and many candidate genes and variants that may be useful for forest tree breeding with special aims. Our findings highlight the importance of integrating genomic and environmental data to predict adaptive capacity of a key forest to rapid climate change in the future.

PtoWRKY40 interacts with PtoPHR1-LIKE3 while regulating the phosphate starvation response in poplar.

Plants usually suffer from phosphorus starvation because of the low inorganic phosphate (Pi) status of most soils. To cope with this, plants have evolved an adaptive phosphate starvation response (PSR) which involves both developmental and metabolic changes regulated mainly by PHOSPHATE STARVATION RESPONSE1 (PHR1) and its homologs. Here, we elucidated how perennial woody plants, such as poplars (Populus spp.), respond to low-Pi stress. We first performed RNA-seq analysis of low-Pi-treated poplars and identified PtoWRKY40 is rapidly downregulated and protein degraded after stress. Overexpressing and knocking-down PtoWRKY40 downregulated and upregulated the expression of Pi starvation signaling genes, respectively, such as PHOSPHATE TRANSPORTER1 (PHT1)-type genes and PURPLE ACID PHOSPHATASE genes. PtoWRKY40 bound to the W box in the promoter of several PtoPHT1s and repressed their expression. Moreover, PtoWRKY40 interacted with PtoPHR1-LIKE3 (PtoPHL3), a PHR1 homolog in poplar, to inhibit the latter binding to the P1BS element and thus reduced PtoPHT1s' transcription under Pi-sufficient conditions. However, Pi deficiency decreased PtoWRKY40 abundance and therefore released its inhibition on PHT1s. In conclusion, we have uncovered a PSR mechanism mediated by PtoWRKY40 and PtoPHL3 which regulates Pi content in poplars, deepening our understanding of how poplars adapt to diverse Pi conditions and regulate appropriate responses to maintain Pi homeostasis.

PtoNF-YC9-SRMT-PtoRD26 module regulates the high saline tolerance of a triploid poplar.

BACKGROUND: Sensing and responding to stresses determine the tolerance of plants to adverse environments. The triploid Chinese white poplar is widely cultivated in North China because of its adaptation to a wide range of habitats including highly saline ones. However, its triploid genome complicates any detailed investigation of the molecular mechanisms underlying its adaptations. RESULTS: We report a haplotype-resolved genome of this triploid poplar and characterize, using reverse genetics and biochemical approaches, a MYB gene, SALT RESPONSIVE MYB TRANSCRIPTION FACTOR (SRMT), which combines NUCLEAR FACTOR Y SUBUNIT C 9 (PtoNF-YC9) and RESPONSIVE TO DESICCATION 26 (PtoRD26), to regulate an ABA-dependent salt-stress response signaling. We reveal that the salt-inducible PtoRD26 is dependent on ABA signaling. We demonstrate that ABA or salt drives PtoNF-YC9 shuttling into the nucleus where it interacts with SRMT, resulting in the rapid expression of PtoRD26 which in turn directly regulates SRMT. This positive feedback loop of SRMT-PtoRD26 can rapidly amplify salt-stress signaling. Interference with either component of this regulatory module reduces the salt tolerance of this triploid poplar. CONCLUSION: Our findings reveal a novel ABA-dependent salt-responsive mechanism, which is mediated by the PtoNF-YC9-SRMT-PtoRD26 module that confers salt tolerance to this triploid poplar. These genes may therefore also serve as potential and important modification targets in breeding programs.

One AP2/ERF Transcription Factor Positively Regulates Pi Uptake and Drought Tolerance in Poplar.

Drought decreases the inorganic phosphate (Pi) supply of soil, resulting in Pi starvation of plants, but the molecular mechanism of how plants, especially the perennial trees, are tolerant to drought stress and Pi starvation, is still elusive. In this study, we identified an AP2/ERF transcription factor gene, PalERF2, from Populus alba var. pyramidalis, and it was induced by both mannitol treatment and Pi starvation. Overexpressing and knocking-down of PalERF2 both enhanced and attenuated tolerance to drought stress and Pi deficiency compared to WT, respectively. Moreover, the overexpression of PalERF2 up-regulated the expression levels of Pi starvation-induced (PSI) genes and increased Pi uptake under drought conditions; however, its RNAi poplar showed the opposite phenotypes. Subsequent analysis indicated that PalERF2 directly modulated expressions of drought-responsive genes PalRD20 and PalSAG113, as well as PSI genes PalPHL2 and PalPHT1;4, through binding to the DRE motifs on their promoters. These results clearly indicate that poplars can recruit PalERF2 to increase the tolerance to drought and also elevate Pi uptake under drought stress.

Molecular signatures of parallel adaptive divergence causing reproductive isolation and speciation across two genera.

Parallel evolution of reproductive isolation (PERI) provides strong evidence for natural selection playing a fundamental role in the origin of species. However, PERI has been rarely demonstrated for well established species drawn from different genera. In particular, parallel molecular signatures for the same genes in response to similar habitat divergence in such different lineages is lacking. Here, based on whole-genome sequencing data, we first explore the speciation process in two sister species of Carpinus (Betulaceae) in response to divergence for temperature and soil-iron concentration in habitats they occupy in northern and southwestern China, respectively. We then determine whether parallel molecular mutations occur during speciation in this pair of species and also in another sister-species pair of the related genus, Ostryopsis, which occupy similarly divergent habitats in China. We show that gene flow occurred during the origin of both pairs of sister species since approximately 9.8 or approximately 2 million years ago, implying strong natural selection during divergence. Also, in both species pairs we detected concurrent positive selection in a gene (LHY) for flowering time and in two paralogous genes (FRO4 and FRO7) of a gene family known to be important for iron tolerance. These changes were in addition to changes in other major genes related to these two traits. The different alleles of these particular candidate genes possessed by the sister species of Carpinus were functionally tested and indicated likely to alter flowering time and iron tolerance as previously demonstrated in the pair of Ostryopsis sister species. Allelic changes in these genes may have effectively resulted in high levels of prezygotic reproductive isolation to evolve between sister species of each pair. Our results show that PERI can occur in different genera at different timescales and involve similar signatures of molecular evolution at genes or paralogues of the same gene family, causing reproductive isolation as a consequence of adaptation to similarly divergent habitats.

Allelic shift in cis-elements of the transcription factor RAP2.12 underlies adaptation associated with humidity in Arabidopsis thaliana.

Populations of widespread species are usually geographically distributed through contrasting stresses, but underlying genetic mechanisms controlling this adaptation remain largely unknown. Here, we show that in Arabidopsis thaliana, allelic changes in the cis-regulatory elements, WT box and W box, in the promoter of a key transcription factor associated with oxygen sensing, RELATED TO AP 2.12 (RAP2.12), are responsible for differentially regulating tolerance to drought and flooding. These two cis-elements are regulated by different transcription factors that downstream of RAP2.12 results in differential accumulation of hypoxia-responsive transcripts. The evolution from one cis-element haplotype to the other is associated with the colonization of humid environments from arid habitats. This gene thus promotes both drought and flooding adaptation via an adaptive mechanism that diversifies its regulation through noncoding alleles.

Pervasive hybridization during evolutionary radiation of Rhododendron subgenus Hymenanthes in mountains of southwest China.

Radiations are especially important for generating species biodiversity in mountainous ecosystems. The contribution of hybridization to such radiations has rarely been examined. Here, we use extensive genomic data to test whether hybridization was involved in evolutionary radiation within Rhododendron subgenus Hymenanthes, whose members show strong geographic isolation in the mountains of southwest China. We sequenced genomes for 143 species of this subgenus and 93 species of four other subgenera, and found that Hymenanthes was monophyletic and radiated during the late Oligocene to middle Miocene. Widespread hybridization events were inferred within and between the identified clades and subclades. This suggests that hybridization occurred both early and late during diversification of subgenus Hymenanthes, although the extent to which hybridization, speciation through mixing-isolation-mixing or hybrid speciation, accelerated the diversification needs further exploration. Cycles of isolation and contact in such and other montane ecosystems may have together promoted species radiation through hybridization between diverging populations and species. Similar radiation processes may apply to other montane floras in this region and elsewhere.

Genome-Wide Analysis of the Homeobox Gene Family and Identification of Drought-Responsive Members in Populus trichocarpa.

Homeobox (HB) genes play critical roles in the regulation of plant morphogenesis, growth and development. Here, we identified a total of 156 PtrHB genes from the Populus trichocarpa genome. According to the topologies and taxonomy of the phylogenetic tree constructed by Arabidopsis thaliana HB members, all PtrHB proteins were divided into six subgroups, namely HD-ZIP, ZF-HD, HB-PHD, TALE, WOX and HB-OTHERS. Multiple alignments of conserved homeodomains (HDs) revealed the conserved loci of each subgroup, while gene structure analysis showed similar exon-intron gene structures, and motif analysis indicated the similarity of motif number and pattern in the same subgroup. Promoter analysis indicated that the promoters of PtrHB genes contain a series of cis-acting regulatory elements involved in responding to various abiotic stresses, indicating that PtrHBs had potential functions in these processes. Collinearity analysis revealed that there are 96 pairs of 127 PtrHB genes mainly distributing on Chromosomes 1, 2, and 5. We analyzed the spatio-temporal expression patterns of PtrHB genes, and the virus-induced gene silencing (VIGS) of PtrHB3 gene resulted in the compromised tolerance of poplar seedlings to mannitol treatment. The bioinformatics on PtrHB family and preliminary exploration of drought-responsive genes can provide support for further study of the family in woody plants, especially in drought-related biological processes. It also provides a direction for developing new varieties of poplar with drought resistance. Overall, our results provided significant information for further functional analysis of PtrHB genes in poplar and demonstrated that PtrHB3 is a dominant gene regulating tolerance to water stress treatment in poplar seedlings.

Transcriptional landscape of highly lignified poplar stems at single-cell resolution.

BACKGROUND: Plant secondary growth depends on the activity of the vascular cambium, which produces xylem and phloem. Wood derived from xylem is the most abundant form of biomass globally and has played key socio-economic and subsistence roles throughout human history. However, despite intensive study of vascular development, the full diversity of cell types and the gene networks engaged are still poorly understood. RESULTS: Here, we have applied an optimized protoplast isolation protocol and RNA sequencing to characterize the high-resolution single-cell transcriptional landscape of highly lignified poplar stems. We identify 20 putative cell clusters with a series of novel cluster-specific marker genes and find that these cells are highly heterogeneous based on the transcriptome. Analysis of these marker genes' expression dynamics enables reconstruction of the cell differentiation trajectories involved in phloem and xylem development. We find that different cell clusters exhibit distinct patterns of phytohormone responses and emphasize the use of our data to predict potential gene redundancy and identify candidate genes related to vascular development in trees. CONCLUSIONS: These findings establish the transcriptional landscape of major cell types of poplar stems at single-cell resolution and provide a valuable resource for investigating basic principles of vascular cell specification and differentiation in trees.

The U-box E3 ubiquitin ligase PalPUB79 positively regulates ABA-dependent drought tolerance via ubiquitination of PalWRKY77 in Populus.

The abscisic acid (ABA) signalling pathway is involved in the plant response to osmotic stress caused by drought and/or salinity. Although the ABA signalling pathway has been elucidated in Arabidopsis, it remains elusive in woody poplars. In this study, genome-wide analyses of U-box genes in poplars revealed that a U-box E3 ubiquitin ligase gene, PalPUB79, is significantly induced following drought, salinity and ABA signalling. PalPUB79 overexpression enhanced drought tolerance in transgenic poplars, while PalPUB79 RNAi lines were more sensitive to drought. PalPUB79 positively regulated ABA signalling pathway. Furthermore, PalPUB79 interacted with PalWRKY77, a negative transcriptional regulator of ABA signalling, and mediated its ubiquitination for degradation, therefore counteracting its inhibitory effect on PalRD26 transcription. However, the finding that PalWRKY77 negatively regulates PalPUB79 expression was indicative of a negative feedback loop between PalWRKY77 and PalPUB79 during ABA signalling in poplar. These findings provide novel insight into the mechanism through which PalPUB79 enhances the ABA-mediated stress response in woody poplars.

The ubiquitin E3 ligase SR1 modulates the submergence response by degrading phosphorylated WRKY33 in Arabidopsis.

Oxygen deprivation caused by flooding activates acclimation responses to stress and restricts plant growth. After experiencing flooding stress, plants must restore normal growth; however, which genes are dynamically and precisely controlled by flooding stress remains largely unknown. Here, we show that the Arabidopsis thaliana ubiquitin E3 ligase SUBMERGENCE RESISTANT1 (SR1) regulates the stability of the transcription factor WRKY33 to modulate the submergence response. SR1 physically interacts with WRKY33 in vivo and in vitro and controls its ubiquitination and proteasomal degradation. Both the sr1 mutant and WRKY33 overexpressors exhibited enhanced submergence tolerance and enhanced expression of hypoxia-responsive genes. Genetic experiments showed that WRKY33 functions downstream of SR1 during the submergence response. Submergence induced the phosphorylation of WRKY33, which enhanced the activation of RAP2.2, a positive regulator of hypoxia-response genes. Phosphorylated WRKY33 and RAP2.2 were degraded by SR1 and the N-degron pathway during reoxygenation, respectively. Taken together, our findings reveal that the on-and-off module SR1-WRKY33-RAP2.2 is connected to the well-known N-degron pathway to regulate acclimation to submergence in Arabidopsis. These two different but related modulation cascades precisely balance submergence acclimation with normal plant growth.

CHYR1 ubiquitinates the phosphorylated WRKY70 for degradation to balance immunity in Arabidopsis thaliana.

It is critically important for plants to control the trade-off between normal growth and pathogen immunity. However, the underlying molecular mechanism remains largely unknown. Here we report such a mechanism controlled by WRKY70 and its partner CHYR1 in Arabidopsis. We found that both levels of the WRKY70 target gene SARD1 and the phosphorylated forms of WRKY70 were increased in WRKY70OE plants upon Pst DC3000 infection. Mechanistically, phosphorylation of WRKY70 at Thr22 and Ser34 occurs, which then activates SARD1 expression through binding to a WT box. Phosphorylated WRKY70 is degraded by 26S proteasome via CHYR1 when resuming normal growth after infection. In addition, nonphosphorylated WRKY70 represses SARD1 expression by binding to both W (inhibitory activity site) and WT (active activity site) boxes. The binding of WRKY70 to alternative cis-elements of SARD1 through a phosphorylation-mediated switch controlled by CHYR1 contributes to modulating the balance between immunity and growth.

Hybrid speciation via inheritance of alternate alleles of parental isolating genes.

It is increasingly realized that homoploid hybrid speciation (HHS), which involves no change in chromosome number, is an important mechanism of speciation. HHS will likely increase in frequency as ecological and geographical barriers between species are continuing to be disrupted by human activities. HHS requires the establishment of reproductive isolation between a hybrid and its parents, but the underlying genes and genetic mechanisms remain largely unknown. In this study, we reveal by integrated approaches that reproductive isolation originates in one homoploid hybrid plant species through the inheritance of alternate alleles at genes that determine parental premating isolation. The parent species of this hybrid species are reproductively isolated by differences in flowering time and survivorship on soils containing high concentrations of iron. We found that the hybrid species inherits alleles of parental isolating major genes related to flowering time from one parent and alleles of major genes related to iron tolerance from the other parent. In this way, it became reproductively isolated from one parent by the difference in flowering time and from the other by habitat adaptation (iron tolerance). These findings and further modeling results suggest that HHS may occur relatively easily via the inheritance of alternate parental premating isolating genes and barriers.

The PalWRKY77 transcription factor negatively regulates salt tolerance and abscisic acid signaling in Populus.

High salinity, one of the most widespread abiotic stresses, inhibits photosynthesis, reduces vegetation growth, blocks respiration and disrupts metabolism in plants. In order to survive their long-term lifecycle, trees, such as Populus species, recruit the abscisic acid (ABA) signaling pathway to adapt to a saline environment. However, the molecular mechanism behind the ABA-mediated salt stress response in woody plants remains elusive. We have isolated a WRKY transcription factor gene, PalWRKY77, from Populus alba var. pyramidalis (poplar), the expression of which is repressed by salt stress. PalWRKY77 decreases salt tolerance in poplar. Furthermore, PalWRKY77 negatively regulated ABA-responsive genes and relieved ABA-mediated growth inhibition, indicating that PalWRKY77 is a repressor of the ABA response. In vivo and in vitro assays revealed that PalWRKY77 targets the ABA- and salt-induced PalNAC002 and PalRD26 genes by binding to the W-boxes in their promoters. In addition, overexpression of both PalNAC002 and PalRD26 could elevate salt tolerance in transgenic poplars. These findings reveal a novel negative regulation mechanism for the ABA signaling pathway mediated by PalWRKY77 that results in more sensitivity to salt stress in poplar. This deepens our understanding of the complex responses of woody species to salt stress.

WRKY33 interacts with WRKY12 protein to up-regulate RAP2.2 during submergence induced hypoxia response in Arabidopsis thaliana.

Tolerance of hypoxia is essential for most plants, but the underlying mechanisms are largely unknown. Here we show that adaptation to submergence induced hypoxia in Arabidopsis involves up-regulation of RAP2.2 through interactive action of WRKY33 and WRKY12. WRKY33- or WRKY12-overexpressing plants showed enhanced resistance to hypoxia. Y2H, BiFC, Co-IP and pull-down experiments confirmed the interaction of WRKY33 with WRKY12. Genetic experiments showed that RAP2.2 acts downstream of WRKY33/WRKY12. WRKY33 and WRKY12 can bind to and activate RAP2.2 individually. Genetic and molecular experiments demonstrate that the two WRKYs can synergistically enhance activation towards RAP2.2 to increase hypoxia tolerance. WRKY33 expression is increased in RAP2.2-overexpressing plants, indicating a feedback regulation by RAP2.2 during submergence process, which was corroborated by EMSA, ChIP, dual-LUC and genetic experiments. Our results show that a regulatory cascade module involving WRKY33, WRKY12 and RAP2.2 plays a key role in submergence induced hypoxia response of Arabidopsis and illuminate functions of WRKYs in hypoxia tolerance.

Heterologous Expression of Poplar WRKY18/35 Paralogs in Arabidopsis Reveals Their Antagonistic Regulation on Pathogen Resistance and Abiotic Stress Tolerance via Variable Hormonal Pathways.

WRKY transcription factors (WRKY TFs) are one of the largest protein families in plants, and most of them play vital roles in response to biotic and abiotic stresses by regulating related signaling pathways. In this study, we isolated two WRKY TF genes PtrWRKY18 and PtrWRKY35 from Populustrichocarpa and overexpressed them in Arabidopsis. Expression pattern analyses showed that PtrWRKY18 and PtrWRKY35 respond to salicylic acid (SA), methyl JA (MeJA), abscisic acid (ABA), B. cinereal, and P. syringae treatment. The transgenic plants conferred higher B. cinerea tolerance than wild-type (WT) plants, and real-time quantitative (qRT)-PCR assays showed that PR3 and PDF1.2 had higher expression levels in transgenic plants, which was consistent with their tolerance to B. cinereal. The transgenic plants showed lower P. syringae tolerance than WT plants, and qRT-PCR analysis (PR1, PR2, and NPR1) also corresponded to this phenotype. Germination rate and root analysis showed that the transgenic plants are less sensitive to ABA, which leads to the reduced tolerance to osmotic stress and the increase of the death ratio and stomatal aperture. Compared with WT plants, a series of ABA-related genes (RD29A, ABO3, ABI4, ABI5, and DREB1A) were significantly down-regulated in PtrWRKY18 and PtrWRKY35 overexpression plants. All of these results demonstrated that the two WRKY TFs are multifunctional transcription factors in plant resistance.