Comparative genomic analysis of the Growth-Regulating Factors-Interacting Factors (GIFs) in six Salicaceae species and functional analysis of PeGIF3 reveals their regulatory role in Populus heteromorphic leaves.

BACKGROUND: The growth-regulating factor-interacting factor (GIF) gene family plays a vital role in regulating plant growth and development, particularly in controlling leaf, seed, and root meristem homeostasis. However, the regulatory mechanism of heteromorphic leaves by GIF genes in Populus euphratica as an important adaptative trait of heteromorphic leaves in response to desert environment remains unknown. RESULTS: This study aimed to identify and characterize the GIF genes in P. euphratica and other five Salicaceae species to investigate their role in regulating heteromorphic leaf development. A total of 27 GIF genes were identified and characterized across six Salicaceae species (P. euphratica, Populus pruinose, Populus deltoides, Populus trichocarpa, Salix sinopurpurea, and Salix suchowensis) at the genome-wide level. Comparative genomic analysis among these species suggested that the expansion of GIFs may be derived from the specific Salicaceae whole-genome duplication event after their divergence from Arabidopsis thaliana. Furthermore, the expression data of PeGIFs in heteromorphic leaves, combined with functional information on GIF genes in Arabidopsis, indicated the role of PeGIFs in regulating the leaf development of P. euphratica, especially PeGIFs containing several cis-acting elements associated with plant growth and development. By heterologous expression of the PeGIF3 gene in wild-type plants (Col-0) and atgif1 mutant of A. thaliana, a significant difference in leaf expansion along the medial-lateral axis, and an increased number of leaf cells, were observed between the overexpressed plants and the wild type. CONCLUSION: PeGIF3 enhances leaf cell proliferation, thereby resulting in the expansion of the central-lateral region of the leaf. The findings not only provide global insights into the evolutionary features of Salicaceae GIFs but also reveal the regulatory mechanism of PeGIF3 in heteromorphic leaves of P. euphratica.

Curtobacterium salicis sp. nov., isolated from willow tree stems in Washington state.

Curtobacterium sp. strain WW7 is a Gram-positive, non-motile, orange rod-shaped bacterium isolated from branches of wild willow (Salix sitchensis) trees. The WW7T strain has optimum growth in the temperature range between 25 and 30 °C, a pH range of 6-7.7, and tolerates up to 5.5% (w/v) of NaCl. The genome sequencing of strain WW7T revealed a genome size of approximately 3.8 Mbp and a G + C content of 71.3 mol%. The phylogenomic analyses support the WW7T affiliation to a novel Curtobacterium lineage, with Curtobacterium herbarum being the closest type-strain. Chemotaxonomic analysis indicates that the carbon sources assimilation profile of strain WW7T was similar to the type strains, i.e. Curtobacterium luteum, Curtobacterium albidum, and Curtobacterium flaccumfaciens, while no assimilation of the organic acids succinate, alpha-ketobutyrate, mono methyl-succinate, and lactate was observed. Finally, fatty acid methyl ester analysis identifies anteiso-C15:0 and anteiso-C17:0 as major cellular fatty acids which is a common feature for members of the Curtobacterium genus. Based on the results of phylogenomic and chemotaxonomic analyses, strain WW7T represents a novel Curtobacterium lineage, for which the name Curtobacterium salicis sp. nov. is proposed. The type strain is WW7T (DSM 34805T-NRRL B-68078T).

Characterization, expression pattern, and function analysis of gibberellin oxidases in Salix matsudana.

Gibberellin oxidases (GAoxs) identified from many species play indispensable roles in GA biosynthesis and GA signal transduction. However, there has been limited research conducted on the GAox family of Salix matsudana, a tetraploid ornamental tree species. Here, 54 GAox genes were identified from S. matsudana and renamed as SmGA20ox1-22, SmGA2ox1-24, SmGA3ox1-6, and SmGAox-like1/2. Gene structure and conserved motif analysis showed that SmGA3ox members possess the 1 intron and other SmGAoxs contain 2-3 introns, and motif 1/2/7 universally present in all SmGAoxs. A total of 69 gene pairs were identified from SmGAox family members, and the Ka/Ks values indicated the SmGAoxs experience the purifying selection. The intra species collinearity analysis implied S. matsudana, S. purpurea, and Populus trichocarpa have the close genetic relationship. The GO analysis suggested SmGAoxs are dominantly involved in GA metabolic process, ion binding, and oxidoreductase activity. RNA-sequencing demonstrated that some SmGAoxs may play an essential role in salt and submergence stresses. In addition, the SmGA20ox13/21 displayed the dominant vitality of GA20 oxidase, but the SmGA20ox13/21 still possessed low activities of GA2 and GA3 oxidases. This study can contribute to reveal the regulatory mechanism of salt and submergence tolerance in willow.

Impact of Heavy Metals on Cold Acclimation of Salix viminalis Roots.

In nature, plants are exposed to a range of climatic conditions. Those negatively impacting plant growth and survival are called abiotic stresses. Although abiotic stresses have been extensively studied separately, little is known about their interactions. Here, we investigate the impact of long-term mild metal exposure on the cold acclimation of Salix viminalis roots using physiological, transcriptomic, and proteomic approaches. We found that, while metal exposure significantly affected plant morphology and physiology, it did not impede cold acclimation. Cold acclimation alone increased glutathione content and glutathione reductase activity. It also resulted in the increase in transcripts and proteins belonging to the heat-shock proteins and related to the energy metabolism. Exposure to metals decreased antioxidant capacity but increased catalase and superoxide dismutase activity. It also resulted in the overexpression of transcripts and proteins related to metal homeostasis, protein folding, and the antioxidant machinery. The simultaneous exposure to both stressors resulted in effects that were not the simple addition of the effects of both stressors taken separately. At the antioxidant level, the response to both stressors was like the response to metals alone. While this should have led to a reduction of frost tolerance, this was not observed. The impact of the simultaneous exposure to metals and cold acclimation on the transcriptome was unique, while at the proteomic level the cold acclimation component seemed to be dominant. Some genes and proteins displayed positive interaction patterns. These genes and proteins were related to the mitigation and reparation of oxidative damage, sugar catabolism, and the production of lignans, trehalose, and raffinose. Interestingly, none of these genes and proteins belonged to the traditional ROS homeostasis system. These results highlight the importance of the under-studied role of lignans and the ROS damage repair and removal system in plants simultaneously exposed to multiple stressors.

Comprehensive bioinformation analysis of homeodomain-leucine zipper gene family and expression pattern of HD-Zip I under abiotic stress in Salix suchowensis.

BACKGROUND: Homeodomain-leucine zipper (HD-Zip) transcription factors are plant-specific and play important roles in plant defense against environmental stresses. Identification and functional studies have been carried out in model plants such as rice, Arabidopsis thaliana, and poplar, but comprehensive analysis on the HD-Zip family of Salix suchowensis have not been reported. RESULTS: A total of 55 HD-Zip genes were identified in the willow genome, unevenly distributed on 18 chromosomes except for chromosome 19. And segmental duplication events containing SsHD-Zip were detected on all chromosomes except chromosomes 13 and 19. The SsHD-Zip were classified into 4 subfamilies subfamilies (I-IV) according to the evolutionary analysis, and members of each subfamily shared similar domain structure and gene structure. The combination of GO annotation and promoter analysis showed that SsHD-Zip genes responded to multiple abiotic stresses. Furthermore, the results of qPCR analysis showed that the SsHD-Zip I gene exhibited different degrees of expression under salt stress, PEG treatment and heat treatment. Moreover, there was a synergistic effect between SsHD-Zip I genes under stress conditions based on coregulatory networks analysis. CONCLUSIONS: In this study, HD-Zip transcription factors were systematically identified and analyzed at the whole genome level. These results preliminarily clarified the structural characteristics and related functions of willow HD-Zip family members, and it was found that SsHox34, SsHox36 and SsHox51 genes were significantly involved in the response to various stresses. Together, these findings laid the foundation for further research on the resistance functions of willow HD-Zip genes.

Na2CO3-responsive mechanism insight from quantitative proteomics and SlRUB gene function in Salix linearistipularis seedlings.

Mongolian willow (Salix linearistipularis) is a naturally occurring woody dioecious plant in the saline soils of north-eastern China, which has a high tolerance to alkaline salts. Although transcriptomics studies have identified a large number of salinity-responsive genes, the mechanism of salt tolerance in Mongolian willow is not clear. Here, we found that in response to Na2CO3 stress, Mongolian willow regulates osmotic homeostasis by accumulating proline and soluble sugars and scavenges reactive oxygen species (ROS) by antioxidant enzymes and non-enzymatic antioxidants. Our quantitative proteomics study identified 154 salt-sensitive proteins mainly involved in maintaining the stability of the photosynthetic system and ROS homeostasis to cope with Na2CO3 stress. Among them, Na2CO3-induced rubredoxin (RUB) was predicted to be associated with 122 proteins for the modulation of these processes. The chloroplast-localized S. linearistipularis rubredoxin (SlRUB) was highly expressed in leaves and was significantly induced under Na2CO3 stress. Phenotypic analysis of overexpression, mutation and complementation materials of RUB in Arabidopsis suggests that SlRUB is critical for the regulation of photosynthesis, ROS scavenging and other metabolisms in the seedlings of Mongolian willow to cope with Na2CO3 stress. This provides more clues to better understand the alkali-responsive mechanism and RUB functions in the woody Mongolian willow.

Cell number regulator 8 from Salix linearistipularis enhances cadmium tolerance in poplar by reducing cadmium uptake and accumulation.

Trace metals have relatively high density and high toxicity at low concentrations. Willow (Salix genus) is an excellent phytoremediation species for soil contaminated by trace metal ions. This study identified a cell number regulator (CNR) gene family member in Salix linearistipularis exhibiting strong metal ion resistance: SlCNR8. SlCNR8 expression was affected by various metal ions, including cadmium (Cd), zinc (Zn), copper (Cu), iron (Fe), and manganese (Mn). SlCNR8 overexpression enhanced Cd, Zn, Cu, and Fe resistance in transgenic poplar seedlings (84K) compared with the wild-type (WT). Moreover, transgenic poplar seedlings showed lower root Cd uptake and less Cd accumulation than WT under Cd stress. SlCNR8 was primarily localized to the nucleus and the plasma membrane-like cell periphery. Furthermore, SlCNR8 had transcriptional activation activity in yeast. The transcript levels of multiple metal ion transporters were altered in the roots of transgenic poplar seedlings compared to WT roots under Cd stress. These results suggest that SlCNR8 may enhance Cd resistance in transgenic poplar by reducing Cd uptake and accumulation. This may be related to altered transcription levels of other transporters or to itself. Our study suggests that SlCNR8 can be used as a candidate gene for genetic improvement of phytostabilisation of trace metals by genetic engineering.

Genome-wide identification and expression analysis of xyloglucan endotransglucosylase/hydrolase genes family in Salicaceae during grafting.

BACKGROUND: Poplar (Populus cathayana)and willow (Salix rehderiana) are important fast-growing trees in China. Grafting plays an important role in improving plant stress resistance and construction of ornamental plants. It is found that willow scions grafted onto poplar rootstocks can form ornamental plants. However, this grafted combination has a low survival rate. Many studies have reported that the xyloglucan endotransglucosylase/hydrolase (XTH) family plays an important role in the healing process of grafts. RESULTS: A total of 38 PtrXTHs and 32 SpuXTHs were identified in poplar and willow respectively, and were classified into three subfamilies. Tandem duplication was the main reason for the expansion of the PtrXTHs. Grafting treatment and Quantitative real time PCR (RT-qPCR) analysis revealed that five XTH genes differentially expressed between self-grafted and reciprocal grafted combinations. Specifically, the high expression levels of SrXTH16, SrXTH17, SrXTH25, PcXTH22 and PcXTH17 may contribute to the high survival rate of the grafted combination with willow scion and poplar rootstock. Subcellular localization identified that the SrXTH16, SrXTH17, SrXTH25, PcXTH17 and PcXTH22 proteins were located on the cell walls. Transcription factors (NAC, MYB and DOF) may regulate the five XTH genes. CONCLUSIONS: This study provides a new understanding of the roles of PcXTH and SrXTH genes and their roles in grafting. Our results will give some hints to explore the molecular mechanisms of PcXTH and SrXTH genes involved in grafting in the future.

Evolution of a ZW sex chromosome system in willows.

Transitions in the heterogamety of sex chromosomes (e.g., XY to ZW or vice versa) fundamentally alter the genetic basis of sex determination, however the details of these changes have been studied in only a few cases. In an XY to ZW transition, the X is likely to give rise to the W because they both carry feminizing genes and the X is expected to harbour less genetic load than the Y. Here, using a new reference genome for Salix exigua, we trace the X, Y, Z, and W sex determination regions during the homologous transition from an XY system to a ZW system in willow (Salix). We show that both the W and the Z arose from the Y chromosome. We find that the new Z chromosome shares multiple homologous putative masculinizing factors with the ancestral Y, whereas the new W lost these masculinizing factors and gained feminizing factors. The origination of both the W and Z from the Y was permitted by an unexpectedly low genetic load on the Y and this indicates that the origins of sex chromosomes during homologous transitions may be more flexible than previously considered.

Manifestation of Triploid Heterosis in the Root System after Crossing Diploid and Autotetraploid Energy Willow Plants.

Successful use of woody species in reducing climatic and environmental risks of energy shortage and spreading pollution requires deeper understanding of the physiological functions controlling biomass productivity and phytoremediation efficiency. Targets in the breeding of energy willow include the size and the functionality of the root system. For the combination of polyploidy and heterosis, we have generated triploid hybrids (THs) of energy willow by crossing autotetraploid willow plants with leading cultivars (Tordis and Inger). These novel Salix genotypes (TH3/12, TH17/17, TH21/2) have provided a unique experimental material for characterization of Mid-Parent Heterosis (MPH) in various root traits. Using a root phenotyping platform, we detected heterosis (TH3/12: MPH 43.99%; TH21/2: MPH 26.93%) in the size of the root system in soil. Triploid heterosis was also recorded in the fresh root weights, but it was less pronounced (MPH%: 9.63-19.31). In agreement with root growth characteristics in soil, the TH3/12 hybrids showed considerable heterosis (MPH: 70.08%) under in vitro conditions. Confocal microscopy-based imaging and quantitative analysis of root parenchyma cells at the division-elongation transition zone showed increased average cell diameter as a sign of cellular heterosis in plants from TH17/17 and TH21/2 triploid lines. Analysis of the hormonal background revealed that the auxin level was seven times higher than the total cytokinin contents in root tips of parental Tordis plants. In triploid hybrids, the auxin-cytokinin ratios were considerably reduced in TH3/12 and TH17/17 roots. In particular, the contents of cytokinin precursor, such as isopentenyl adenosine monophosphate, were elevated in all three triploid hybrids. Heterosis was also recorded in the amounts of active gibberellin precursor, GA19, in roots of TH3/12 plants. The presented experimental findings highlight the physiological basics of triploid heterosis in energy willow roots.

Transcriptome-Wide Identification and Response Pattern Analysis of the Salix integra NAC Transcription Factor in Response to Pb Stress.

The NAC (NAM-ATAF1/2-CUC) transcription factor family is one of the largest plant-specific transcription factor families, playing an important role in plant growth and development and abiotic stress response. As a short-rotation woody plant, Salix integra (S. integra) has high lead (Pb) phytoremediation potential. To understand the role of NAC in S. integra Pb tolerance, 53 SiNAC transcripts were identified using third-generation and next-generation transcriptomic data from S. integra exposed to Pb stress, and a phylogenetic analysis revealed 11 subfamilies. A sequence alignment showed that multiple subfamilies represented by TIP and ATAF had a gene that produced more than one transcript under Pb stress, and different transcripts had different responses to Pb. By analyzing the expression profiles of SiNACs at 9 Pb stress time points, 41 of 53 SiNACs were found to be significantly responsive to Pb. Short time-series expression miner (STEM) analysis revealed that 41 SiNACs had two significant Pb positive response patterns (early and late), both containing 10 SiNACs. The SiNACs with the most significant Pb response were mainly from the ATAF and NAP subfamilies. Therefore, 4 and 3 SiNACs from the ATAF and NAP subfamilies, respectively, were selected as candidate Pb-responsive SiNACs for further structural and functional analysis. The RT-qPCR results of 7 transcripts also confirmed the different Pb response patterns of the ATAF and NAP subfamilies. SiNAC004 and SiNAC120, which were randomly selected from two subfamilies, were confirmed to be nuclear localization proteins by subcellular localization experiments. Functional prediction analysis of the associated transcripts of seven candidate SiNACs showed that the target pathways of ATAF subfamily SiNACs were "sulfur metabolism" and "glutathione metabolism", and the target pathways of NAP subfamily SiNACs were "ribosome" and "phenylpropanoid biosynthesis". This study not only identified two NAC subfamilies with different Pb response patterns but also identified Pb-responsive SiNACs that could provide a basis for subsequent gene function verification.

Phylogenomics reveals patterns of ancient hybridization and differential diversification that contribute to phylogenetic conflict in willows, poplars, and close relatives.

Despite the economic, ecological, and scientific importance of the genera Salix L. (willows) and Populus L. (poplars, cottonwoods, and aspens) Salicaceae, we know little about the sources of differences in species diversity between the genera and of the phylogenetic conflict that often confounds estimating phylogenetic trees. Salix subgenera and sections, in particular, have been difficult to classify, with one recent attempt termed a "spectacular failure" due to a speculated radiation of the subgenera Vetrix and Chamaetia. Here, we use targeted sequence capture to understand the evolutionary history of this portion of the Salicaceae plant family. Our phylogenetic hypothesis was based on 787 gene regions and identified extensive phylogenetic conflict among genes. Our analysis supported some previously described subgeneric relationships and confirmed the polyphyly of others. Using an fbranch analysis, we identified several cases of hybridization in deep branches of the phylogeny, which likely contributed to discordance among gene trees. In addition, we identified a rapid increase in diversification rate near the origination of the Vetrix-Chamaetia clade in Salix. This region of the tree coincided with several nodes that lacked strong statistical support, indicating a possible increase in incomplete lineage sorting due to rapid diversification. The extraordinary level of both recent and ancient hybridization in both Salix and Populus have played important roles in the diversification and diversity in these two genera.

Contrasting levels of ß-diversity and underlying phylogenetic trends indicate different paths to chemical diversity in highland and lowland willow species.

Diverse specialised metabolites contributed to the success of vascular plants in colonising most terrestrial habitats. Understanding how distinct aspects of chemical diversity arise through heterogeneous environmental pressures can help us understand the effects of abiotic and biotic stress on plant evolution and community assembly. We examined highland and lowland willow species within a phylogenetic framework to test for trends in their chemical α-diversity (richness) and ß-diversity (variation among species sympatric in elevation). We show that differences in chemistry among willows growing at different elevations occur mainly through shifts in chemical ß-diversity and due to convergence or divergence among species sharing their elevation level. We also detect contrasting phylogenetic trends in concentration and α-diversity of metabolites in highland and lowland willow species. The resulting elevational patterns contribute to the chemical diversity of willows and suggest that variable selective pressure across ecological gradients may, more generally, underpin complex changes in plant chemistry.

A chromosome-scale genome assembly and evaluation of mtDNA variation in the willow leaf beetle Chrysomela aeneicollis.

The leaf beetle Chrysomela aeneicollis has a broad geographic range across Western North America but is restricted to cool habitats at high elevations along the west coast. Central California populations occur only at high altitudes (2,700-3,500 m) where they are limited by reduced oxygen supply and recent drought conditions that are associated with climate change. Here, we report a chromosome-scale genome assembly alongside a complete mitochondrial genome and characterize differences among mitochondrial genomes along a latitudinal gradient over which beetles show substantial population structure and adaptation to fluctuating temperatures. Our scaffolded genome assembly consists of 21 linkage groups; one of which we identified as the X chromosome based on female/male whole genome sequencing coverage and orthology with Tribolium castaneum. We identified repetitive sequences in the genome and found them to be broadly distributed across all linkage groups. Using a reference transcriptome, we annotated a total of 12,586 protein-coding genes. We also describe differences in putative secondary structures of mitochondrial RNA molecules, which may generate functional differences important in adaptation to harsh abiotic conditions. We document substitutions at mitochondrial tRNA molecules and substitutions and insertions in the 16S rRNA region that could affect intermolecular interactions with products from the nuclear genome. This first chromosome-level reference genome will enable genomic research in this important model organism for understanding the biological impacts of climate change on montane insects.

Structural variation of a sex-linked region confers monoecy and implicates GATA15 as a master regulator of sex in Salix purpurea.

The Salicaceae, including Populus and Salix, are dioecious perennials that utilize different sex determination systems. This family provides a useful system to better understand the evolution of dioecy and sex chromosomes. Here, a rare monoecious genotype of Salix purpurea, 94003, was self- and cross-pollinated and progeny sex ratios were used to test hypotheses on possible mechanisms of sex determination. To delimit genomic regions associated with monoecious expression, the 94003 genome sequence was assembled and DNA- and RNA-Seq of progeny inflorescences was performed. Based on alignments of progeny shotgun DNA sequences to the haplotype-resolved monoecious 94003 genome assembly and reference male and female genomes, a 1.15 Mb sex-linked region on Chr15W was confirmed to be absent in monecious plants. Inheritance of this structural variation is responsible for the loss of a male-suppressing function in what would otherwise be genetic females (ZW), resulting in monoecy (ZWH or WWH ), or lethality, if homozygous (WH WH ). We present a refined, two-gene sex determination model for Salix purpurea, mediated by ARR17 and GATA15 that is different from the single-gene ARR17-mediated system in the related genus Populus.

De Novo Assembly and Annotation of 11 Diverse Shrub Willow (Salix) Genomes Reveals Novel Gene Organization in Sex-Linked Regions.

Poplar and willow species in the Salicaceae are dioecious, yet have been shown to use different sex determination systems located on different chromosomes. Willows in the subgenus Vetrix are interesting for comparative studies of sex determination systems, yet genomic resources for these species are still quite limited. Only a few annotated reference genome assemblies are available, despite many species in use in breeding programs. Here we present de novo assemblies and annotations of 11 shrub willow genomes from six species. Copy number variation of candidate sex determination genes within each genome was characterized and revealed remarkable differences in putative master regulator gene duplication and deletion. We also analyzed copy number and expression of candidate genes involved in floral secondary metabolism, and identified substantial variation across genotypes, which can be used for parental selection in breeding programs. Lastly, we report on a genotype that produces only female descendants and identified gene presence/absence variation in the mitochondrial genome that may be responsible for this unusual inheritance.

Accumulation of retrotransposons contributes to W chromosome differentiation in the willow beauty Peribatodes rhomboidaria (Lepidoptera: Geometridae).

The W chromosome of Lepidoptera is typically gene-poor, repeat-rich and composed of heterochromatin. Pioneering studies investigating this chromosome reported an abundance of mobile elements. However, the actual composition of the W chromosome varies greatly between species, as repeatedly demonstrated by comparative genomic hybridization (CGH) or fluorescence in situ hybridization (FISH). Here we present an analysis of repeats on the W chromosome in the willow beauty, Peribatodes rhomboidaria (Geometridae), a species in which CGH predicted an abundance of W-enriched or W-specific sequences. Indeed, comparative analysis of male and female genomes using RepeatExplorer identified ten putative W chromosome-enriched repeats, most of which are LTR or LINE mobile elements. We analysed the two most abundant: PRW LINE-like and PRW Bel-Pao. The results of FISH mapping and bioinformatic analysis confirmed their enrichment on the W chromosome, supporting the hypothesis that mobile elements are the driving force of W chromosome differentiation in Lepidoptera. As the W chromosome is highly underrepresented in chromosome-level genome assemblies of Lepidoptera, this recently introduced approach, combining bioinformatic comparative genome analysis with molecular cytogenetics, provides an elegant tool for studying this elusive and rapidly evolving part of the genome.

The male-heterogametic sex determination system on chromosome 15 of Salix triandra and Salix arbutifolia reveals ancestral male heterogamety and subsequent turnover events in the genus Salix.

Dioecious Salix evolved more than 45 million years ago, but have homomorphic sex chromosomes, suggesting that turnover event(s) prevented major differentiation. Sex chromosome turnover events have been inferred in the sister genus Populus. The genus Salix includes two main clades, Salix and Vetrix, with several previously studied Vetrix clade species having female-heterogametic (ZW) or male-heterogametic (XY) sex-determining systems (SDSs) on chromosome 15, while three Salix clade species have XY SDSs on chromosome 7. We here studied two basal taxa of the Vetrix clade, S. arbutifolia and S. triandra using S. purpurea as the reference genome. Analyses of whole genome resequencing data for genome-wide associations (GWAS) with the sexes and genetic differentiation between the sexes (FST values) showed that both species have male heterogamety with a sex-determining locus on chromosome 15, suggesting an early turnover event within the Vetrix clade, perhaps promoted by sexually antagonistic or (and) sex-ratio selection. Changepoint analysis based on FST values identified small sex-linked regions of ~3.33 Mb and ~2.80 Mb in S. arbutifolia and S. triandra, respectively. The SDS of S. arbutifolia was consistent with recent results that used its own genome as reference. Ancestral state reconstruction of SDS suggests that at least two turnover events occurred in Salix.

Cloning and Functional Identification of Gibberellin Receptor SvGID1s Gene of Salix viminalis.

Gibberellins (GAs) play a key role in the transition from vegetative growth to flowering and the GA receptor GID1 (GIBBERELLIN INSENSITIVE DWARF1) is the central part of GA-signaling. The differential expression of SvGID1 was found in the transcriptome sequencing in our previous study, which was further verified at different stages of flowering of Salix viminalis. In order to reveal the function GID1 of S. viminalis, two genes of SvGID1b and SvGID1c were cloned and transformed into Arabidopsis thaliana, respectively. The results showed that the full ORF length of SvGID1b and SvGID1c genes were both 1035 bp, encoding 344 amino acids, which were typical globular proteins. The peptide chain contained more α-helix structure, and had 99% similarity with GID1b and GID1c amino acid sequences of Salix suchowensis. Phylogenetic analysis showed that SvGID1s had close genetic relationship with woody plants such as Populus alba and Populus tomentosa, and had far genetic relationship with rice. After overexpression in A. thaliana, the total gibberellin, active gibberellin content and the expression level of GA3ox1, the key gene for GA4 synthesis, were not significantly different from those in the wild-type, while the expression levels of FUL, SOC1 and FT, the key genes for flowering in plants, were increased, and the expression levels of FLC and GAI were decreased. The ectopic expression of SvGID1s increased the sensitivity of plants to gibberellin and enhanced gibberellin effect, caused early bolting, budding and flowering, led to higher plant, longer hypocotyl and other phenomena. The results provide a theoretical basis for clarifying the regulation of gibberellin on flower bud differentiation of flowering plants.

Molecular insights into lignin biosynthesis on cadmium tolerance: Morphology, transcriptome and proteome profiling in Salix matsudana.

Soil pollution caused by cadmium (Cd) is a serious concern. Phytoremediation is a popular technology in the remediation of Cd-contaminated soil. Salix matsudana var. matsudana f. umbraculifera Rehd. has been characterized as a high Cd-accumulating and tolerant willow (HCW). Here, transcriptome and proteome profiling, along with morphology analyses were performed to explore molecular cross-talk involved in Cd tolerance. Our results showed that 73%- 83% of the Cd in roots accumulated in the cell walls and root xylem cell walls were significantly thickened. From transcriptome and proteome analysis, a total of 153 up-regulated differentially-expressed genes and 655 up-regulated differentially-expressed proteins were found in common between two comparison groups (1 d and 4 d vs. respective control). Furthermore, phenylpropanoid biosynthesis was identified as a key pathway in response to Cd stress. In this pathway, lignin biosynthesis genes or proteins were significantly up-regulated, and lignin content increased significantly in roots under Cd stress. Two Cd-induced genes cinnamoyl-CoA reductase 1 (SmCCR1) and cinnamyl alcohol dehydrogenase 7 (SmCAD7) from HCW increased the lignin content and enhanced Cd tolerance in transgenic poplar calli. These results lay the foundation for further clarifying the molecular mechanisms of Cd tolerance in woody plants.