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Main Conclusion: Lysine plays an essential role in the growth differences between male and female S. linearistipularis plants under salt stress. Furthermore, SlDHDPS is identified as a vital gene contributing to the differences in saline-alkali tolerance between male and female plants of S. linearistipularis. Soil salinization is a significant problem that severely restricts agricultural production worldwide. High salinity and low nutrient concentrations consequently prevent the growth of most plant species. Salix linearistipularis is the only woody plant (shrub) naturally distributed in the saline-alkali lands of the Songnen Plain in Northeast China, and it is one of the few plants capable of thriving in soils with extremely high salt and alkaline pH (>9.0) levels. However, insufficient attention has been given to the interplay between salt and nitrogen in the growth and development of S. linearistipularis. Here, the male and female plants of S. linearistipularis were subjected to salt stress with nitrogen-starvation or nitrogen-supplement treatments, and it was found that nitrogen significantly affects the difference in salt tolerance between male and female plants, with nitrogen-starvation significantly enhancing the salt stress tolerance of female plants compared to male plants. Transcriptional analyses showed 66 differentially expressed nitrogen-responsive genes in female and male roots, with most of them showing sexual differences in expression patterns under salinity stress. RNA-seq and RT-qPCR analysis demonstrated that six genes had an opposite salt-induced expression pattern in female and male roots. The expression of the 4-hydroxy-tetrahydrodipicolinate synthase encoding gene (SlDHDPS) in female roots was higher than that in male roots. Further treatment with exogenous lysine could significantly alleviate the inhibitory effect of salt stress on the growth of female and male plants. These results indicate that the SlDHDPS in the nitrogen metabolism pathway is involved in the resistance of S. linearistipularis to salt stress, which lays a foundation for further exploring the mechanism of nitrogen on salt tolerance of S. linearistipularis, and has a significant reference value for saline-alkali land management and sustainable agricultural development., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Polyploidization presents an unusual challenge for species with sex chromosomes, as it can lead to complex combinations of sex chromosomes that disrupt reproductive development. This is particularly true for allopolyploidization between species with different sex chromosome systems. Here, we assemble haplotype-resolved chromosome-level genomes of a female allotetraploid weeping willow (Salix babylonica) and a male diploid S. dunnii. We show that weeping willow arose from crosses between a female ancestor from the Salix-clade, which has XY sex chromosomes on chromosome 7, and a male ancestor from the Vetrix-clade, which has ancestral XY sex chromosomes on chromosome 15. We find that weeping willow has one pair of sex chromosomes, ZW on chromosome 15, that derived from the ancestral XY sex chromosomes in the male ancestor of the Vetrix-clade. Moreover, the ancestral 7X chromosomes from the female ancestor of the Salix-clade have reverted to autosomal inheritance. Duplicated intact ARR17-like genes on the four homologous chromosomes 19 likely have contributed to the maintenance of dioecy during polyploidization and sex chromosome turnover. Taken together, our results suggest the rapid evolution and reversion of sex chromosomes following allopolyploidization in weeping willow., (© 2024. The Author(s).)

Premise: The huge diversity of Salix subgenus Chamaetia/Vetrix clade in North America and the lack of phylogenetic resolution within this clade has presented a difficult but fascinating challenge for taxonomists to resolve. Here we tested the existing taxonomic classification with molecular tools., Methods: In this study, 132 samples representing 46 species from 22 described sections of shrub willows from the United States and Canada were analyzed and combined with 67 samples from Eurasia. The ploidy levels of the samples were determined using flow cytometry and nQuire. Sequences were produced using a RAD sequencing approach and subsequently analyzed with ipyrad, then used for phylogenetic reconstructions (RAxML, SplitsTree), dating analyses (BEAST, SNAPPER), and character evolution analyses of 14 selected morphological traits (Mesquite)., Results: The RAD sequencing approach allowed the production of a well-resolved phylogeny of shrub willows. The resulting tree showed an exclusively North American (NA) clade in sister position to a Eurasian clade, which included some North American endemics. The NA clade began to diversify in the Miocene. Polyploid species appeared in each observed clade. Character evolution analyses revealed that adaptive traits such as habit and adaxial nectaries evolved multiple times independently., Conclusions: The diversity in shrub willows was shaped by an evolutionary radiation in North America. Most species were monophyletic, but the existing sectional classification could not be supported by molecular data. Nevertheless, monophyletic lineages share several morphological characters, which might be useful in the revision of the taxonomic classification of shrub willows., (© 2024 The Author(s). American Journal of Botany published by Wiley Periodicals LLC on behalf of Botanical Society of America.)

The Salicaceae includes approximately 54 genera and over 1,400 species with a cosmopolitan distribution. Members of the family are well-known for their diverse secondary plant metabolites, and they play crucial roles in tropical and temperate forest ecosystems. Phylogenetic reconstruction of the Salicaceae has been historically challenging due to the limitations of molecular markers and the extensive history of hybridization and polyploidy within the family. Our study employs whole-genome sequencing of 74 species to generate an extensive phylogeny of the Salicaceae. We generated two RAD-Seq enriched whole-genome sequence datasets and extracted two additional gene sets corresponding to the universal Angiosperms353 and Salicaceae-specific targeted-capture arrays. We reconstructed maximum likelihood-based molecular phylogenies using supermatrix and coalescent-based supertree approaches. Our fossil-calibrated phylogeny estimates that the Salicaceae originated around 128 million years ago and unravels the complex taxonomic relationships within the family. Our findings confirm the non-monophyly of the subgenus Salix s.l. and further support the merging of subgenera Chamaetia and Vetrix, both of which exhibit intricate patterns within and among different sections. Overall, our study not only enhances our understanding of the evolution of the Salicaceae, but also provides valuable insights into the complex relationships within the family., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests that could have influenced the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Phosphorus (P) plays a crucial role in facilitating plant adaptation to cadmium (Cd) stress. However, the molecular mechanisms underlying P-mediated responses to Cd stress in roots remain elusive. This study investigates the effects of P on the growth, physiology, transcriptome, and metabolome of Salix caprea under Cd stress. The results indicate that Cd significantly inhibits plant growth, while sufficient P alleviates this inhibition. Under Cd exposure, P sufficiency resulted in increased Cd accumulation in roots, along with reduced oxidative stress levels (superoxide anion and hydrogen peroxide contents were reduced by 16.8% and 30.1%, respectively). This phenomenon can be attributed to the enhanced activities of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT), as well as increased levels of antioxidants including ascorbic acid (AsA) and flavonoids under sufficient P conditions. A total of 4208 differentially expressed genes (DEGs) and 552 differentially accumulated metabolites (DAMs) were identified in the transcriptomic and metabolomic analyses, with 2596 DEGs and 113 DAMs identified among treatments with different P levels under Cd stress, respectively. Further combined analyses reveal the potential roles of several pathways in P-mediated Cd detoxification, including flavonoid biosynthesis, ascorbate biosynthesis, and plant hormone signal transduction pathways. Notably, sufficient P upregulates the expression of genes including HMA, ZIP, NRAMP and CAX, all predicted to localize to the cell membrane. This may elucidate the heightened Cd accumulation under sufficient P conditions. These findings provide insights into the roles of P in enhancing plant resistance to Cd stress and improving of phytoremediation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

In the Vetrix clade of Salix, a genus of woody flowering plants, sex determination involves chromosome 15, but an XY system has changed to a ZW system. We studied the detailed genetic changes involved. We used genome sequencing, with chromosome conformation capture (Hi-C) and PacBio HiFi reads to assemble chromosome level gap-free X and Y of Salix arbutifolia, and distinguished the haplotypes in the 15X- and 15Y-linked regions, to study the evolutionary history of the sex-linked regions (SLRs). Our sequencing revealed heteromorphism of the X and Y haplotypes of the SLR, with the X-linked region being considerably larger than the corresponding Y region, mainly due to accumulated repetitive sequences and gene duplications. The phylogenies of single-copy orthogroups within the SLRs indicate that S. arbutifolia and Salix purpurea share an ancestral SLR within a repeat-rich region near the chromosome 15 centromere. During the change in heterogamety, the X-linked region changed to a W-linked one, while the Z was derived from the Y., (© 2024 The Authors New Phytologist © 2024 New Phytologist Foundation.)

Expansins, cell wall proteins, play a significant role in plant stress resistance. Our previous study confirmed the expression of the expansin gene SmEXPA13 from Salix matsudana Koidz. enhanced salt tolerance of plants. This report presented an assay that the expression of SmEXPA13 was higher in the salt-resistant willow variety 9901 than in the salt-sensitive variety Yanjiang. In order to understand the possible reasons, a study of the regulation process was conducted. Despite being cloned from both varieties, SmEXPA13 and its promotor showed no significant differences in the structure and sequence. A transcription factor (TF), SmMYB1R1-L, identified through screening the yeast library of willow cDNA, was found to regulate SmEXPA13. Yeast one-hybrid (Y1H) assay confirmed that SmMYB1R1-L could bind to the MYB element at the -520 bp site on the SmEXPA13 promotor. A dual-luciferase reporter assay also demonstrated that SmMYB1R1-L could greatly activate SmEXPA13 expression. The willow calli with over-expression of SmMYB1R1-L exhibited better physiological performance than the wild type under salt stress. Further testing the expression of SmMYB1R1-L displayed it significantly higher in 9901 willow than that in Yanjiang under salt stress. In conclusion, the high accumulation of SmMYB1R1-L in 9901 willow under salt stress led to the high expression of SmEXPA13, resulting in variations in salt stress resistance among willow varieties. The SmMYB1R1-L/SmEXPA13 cascade module in willow offers a new perspective on plant resistance mechanisms., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest or competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Renewable energy resources such as biomass are crucial for a sustainable global society. Trees are a major source of lignocellulosic biomass, which can vary in response to different environmental factors owing to epigenetic regulation, such as DNA C-methylation. To investigate the effects of DNA methylation on plant development and wood formation, and its impacts on gene expression, with a focus on secondary cell wall (SCW)-associated genes, Salix purpurea plantlets were cloned from buds derived from a single hybrid tree for both treatment and control conditions. For the treatment condition, buds were exposed to 50 μM zebularine in vitro and a combined strategy of whole-genome bisulfite sequencing (WGBS) and RNA-seq was employed to examine the methylome and transcriptome profiles of different tissues collected at various time points under both conditions. Transcriptomic and methylome data revealed that most of the promoter and gene body demethylation had no marked effects on the expression profiles of genes. Nevertheless, gene expression tended to decrease with the increased methylation levels of genes with highly methylated promoters. Results indicated that demethylation is less evident in centromeric regions and sex chromosomes. Promoters of secondary cell wall-associated genes, such as 4-coumarate-CoA ligase-like and Rac-like GTP-binding protein RHO, were differentially methylated in the secondary xylem samples collected from two-month potted treated plants compared to control samples. Our results provide novel insights into DNA methylation and gene expression landscapes and a basis for investigating the epigenetic regulation of wood formation in S. purpurea as a model plant for bioenergy species., (© 2024 The Author(s). Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.)

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., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Curtobacterium sp. strain WW7 is a Gram-positive, non-motile, orange rod-shaped bacterium isolated from branches of wild willow (Salix sitchensis) trees. The WW7 T 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 WW7 T revealed a genome size of approximately 3.8 Mbp and a G + C content of 71.3 mol%. The phylogenomic analyses support the WW7 T 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 WW7 T 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-C 15:0 and anteiso-C 17:0 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 WW7 T represents a novel Curtobacterium lineage, for which the name Curtobacterium salicis sp. nov. is proposed. The type strain is WW7 T (DSM 34805 T -NRRL B-68078 T )., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

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., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

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., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

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., (© The Author(s) 2023. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Shrub willows are widely planted for landscaping, soil remediation, and biomass production, due to their rapid growth rates. Identification of regulatory genes in wood formation would provide clues for genetic engineering of willows for improved growth traits on marginal lands. Here, we conducted an expression quantitative trait locus (eQTL) analysis, using a full sibling F 1 population of Salix suchowensis , to explore the genetic mechanisms underlying wood formation. Based on variants identified from simplified genome sequencing and gene expression data from RNA sequencing, 16,487 eQTL blocks controlling 5505 genes were identified, including 2148 cis-eQTLs and 16,480 trans-eQTLs. eQTL hotspots were identified, based on eQTL frequency in genomic windows, revealing one hotspot controlling genes involved in wood formation regulation. Regulatory networks were further constructed, resulting in the identification of key regulatory genes, including three transcription factors ( JAZ1 , HAT22 , MYB36 ) and CLV1 , BAM1 , CYCB2;4 , CDKB2;1 , associated with the proliferation and differentiation activity of cambium cells. The enrichment of genes in plant hormone pathways indicates their critical roles in the regulation of wood formation. Our analyses provide a significant groundwork for a comprehensive understanding of the regulatory network of wood formation in S . suchowensis .

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., (© 2023. The Author(s).)

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., (© 2023. The Author(s).)

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, GA 19 , in roots of TH3/12 plants. The presented experimental findings highlight the physiological basics of triploid heterosis in energy willow roots.

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.

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., Competing Interests: Conflicts of interest statement The author(s) declare no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America.)

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 (ZW H or WW H ), or lethality, if homozygous (W H W H ). 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., (© 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

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 GA 4 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., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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 (F ST 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 F ST 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., (© 2022. The Author(s), under exclusive licence to The Genetics Society.)

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.

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., (© 2023. The Author(s).)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Secondary chemistry often differs between sexes in dioecious plant species, a pattern attributed to its possible role in the evolution and/or maintenance of dioecy. We used GC-MS to measure floral volatiles emitted from, and LC-MS to quantitate non-volatile secondary compounds contained in, female and male Salix purpurea willow catkins from an F2 family. Using the abundance of these chemicals, we then performed quantitative trait locus (QTL) mapping to locate them on the genome, identified biosynthetic candidate genes in the QTL intervals, and examined expression patterns of candidate genes using RNA-seq. Male flowers emitted more total terpenoids than females, but females produced more benzenoids. Male tissue contained greater amounts of phenolic glycosides, but females had more chalcones and flavonoids. A flavonoid pigment and a spermidine derivative were found only in males. Male catkins were almost twice the mass of females. Forty-two QTL were mapped for 25 chemical traits and catkin mass across 16 of the 19 S. purpurea chromosomes. Several candidate genes were identified, including a chalcone isomerase associated with seven compounds. A better understanding of the genetic basis of the sexually dimorphic chemistry of a dioecious species may shed light on how chemically mediated ecological interactions may have helped in the evolution and maintenance of dioecy., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Phytoextraction in phytoremediation is one of the environmentally friendly methods used for restoring soils contaminated by heavy metals (HMs). The screening and identification of HM-resistant plants and their regulatory genes associated with HM ion transport are the key research aims in this field. In this study, a plant cadmium (Cd) resistance (PCR) gene family member, SlPCR6, was identified in roots of Salix linearistipularis, which exhibits strong HM resistance. The results revealed that SlPCR6 expression was induced in S. linearistipularis roots in response to Cd stress. Furthermore, SlPCR6 was mainly localized on the plasma membrane. Compared with the wild type, SlPCR6 overexpression reduced the Cd and copper (Cu) contents in the transgenic poplar (84 K) and increased its Cd and Cu resistance. The roots of transgenic poplar seedlings had lower net Cd and Cu uptake rates than wild type roots. Further investigation revealed that the transcript levels of multiple HM ion transporters were not significantly different between the roots of the wild type and those of the transgenic poplar. These results suggest that SlPCR6 is directly involved in Cd and Cu transport in S. linearistipularis roots. Therefore, SlPCR6 can serve as a candidate gene to improve the phytoextraction of the HMs Cd and Cu through genetic engineering., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Perennial shrub willow are increasingly being promoted in short-rotation coppice systems as biomass feedstocks, for phytoremediation applications, and for the diverse ecosystem services that can accrue. This renewed interest has led to widespread willow cultivation, particularly of non-native varieties. However, Canadian willow species have not been widely adopted and their inherent diversity has not yet been thoroughly investigated. In this study, 324 genotypes of Salix famelica and Salix eriocephala collected from 33 sites of origin were analyzed using 26,016 single nucleotide polymorphisms to reveal patterns of population structure and genetic diversity. Analyses by Bayesian methods and principal component analysis detected five main clusters that appeared to be largely shaped by geoclimatic variables including mean annual precipitation and the number of frost-free days. The overall observed (H O ) and expected (H E ) heterozygosity were 0.126 and 0.179, respectively. An analysis of molecular variance revealed that the highest genetic variation occurred within genotypes (69%), while 8% of the variation existed among clusters and 23% between genotypes within clusters. These findings provide new insights into the extent of genetic variation that exists within native shrub willow species which could be leveraged in pan-Canadian willow breeding programs., (© 2022. The Author(s).)

Salix pseudolasiogyne (Salicaceae) is a willow tree and has been used as a medicinal herb in Korea to treat pain and fever. As a part of an ongoing study to identify bioactive natural products, potential anti-adipogenic compounds were investigated using the ethanol (EtOH) extract of S. pseudolasiogyne twigs. Phytochemical investigation of the EtOH extracts using liquid chromatography-mass spectrometry (LC/MS) led to the separation of two compounds, oregonin ( 1 ) and 2'- O -acetylsalicortin ( 2 ). The structures of the isolates were identified using nuclear magnetic resonance spectroscopy and LC/MS analysis. To the best of our knowledge, it is the first report identifying oregonin ( 1 ) in twigs of S. pseudolasiogyne . Here, we found that the isolated compounds, oregonin ( 1 ) and 2'- O -acetylsalicortin ( 2 ), showed anti-adipogenic effects during 3T3-L1 cell differentiation. Notably, 2'- O -acetylsalicortin ( 2 ), at a concentration of 50 µM, significantly suppressed lipid accumulation. Moreover, the mRNA and protein levels of lipogenic and adipogenic transcription factors were reduced in 2'- O -acetylsalicortin ( 2 )-treated 3T3-L1 cells. Taken together, these results indicate that 2'- O -acetylsalicortin ( 2 ), isolated from S. pseudolasiogyne twigs, has the potential to be applied as a therapeutic agent to effectively control adipocyte differentiation, a critical stage in the progression of obesity.

Background: Salicaceae species have diverse sex determination systems and frequent sex chromosome turnovers. However, compared with poplars, the diversity of sex determination in willows is poorly understood, and little is known about the evolutionary forces driving their turnover. Here, we characterized the sex determination in two Salix species, S. chaenomeloides and S. arbutifolia, which have an XY system on chromosome 7 and 15, respectively., Results: Based on the assemblies of their sex determination regions, we found that the sex determination mechanism of willows may have underlying similarities with poplars, both involving intact and/or partial homologs of a type A cytokinin response regulator (RR) gene. Comparative analyses suggested that at least two sex turnover events have occurred in Salix, one preserving the ancestral pattern of male heterogamety, and the other changing heterogametic sex from XY to ZW, which could be partly explained by the "deleterious mutation load" and "sexually antagonistic selection" theoretical models. We hypothesize that these repeated turnovers keep sex chromosomes of willow species in a perpetually young state, leading to limited degeneration., Conclusions: Our findings further improve the evolutionary trajectory of sex chromosomes in Salicaceae species, explore the evolutionary forces driving the repeated turnovers of their sex chromosomes, and provide a valuable reference for the study of sex chromosomes in other species., (© 2022. The Author(s).)

Background: Gene conversion has an important effect on duplicate genes produced by polyploidization. Poplar (Populus trichocarpa) and willow (Salix brachista) are leading models and excellent green plants in the Salicaceae. Although much attention has been paid to the evolution of duplicated genes in poplar and willow, the role of conversion between duplicates generated from polyploidization remains poorly understood., Results: Here, through genomic synteny analyses, we identified duplicate genes generated by the Salicaceae common tetraploidization (SCT) in the poplar and willow genomes. We estimated that at least 0.58% and 0.25% of poplar and willow duplicates were affected by whole-gene conversion after the poplar-willow divergence, with more (5.73% and 2.66%) affected by partial-gene conversion. Moreover, we found that the converted duplicated genes were unevenly distributed on each chromosome in the two genomes, and the well-preserved homoeologous chromosome regions may facilitate the conversion of duplicates. Notably, we found that conversion maintained the similarity of duplicates, likely contributing to the conservation of certain sequences, but is essentially accelerated the rate of evolution and increased species divergence. In addition, we found that converted duplicates tended to have more similar expression patterns than nonconverted duplicates. We found that genes associated with multigene families were preferentially converted. We also found that the genes encoding conserved structural domains associated with specific traits exhibited a high frequency of conversion., Conclusions: Extensive conversion between duplicate genes generated from the SCT contributes to the diversification of the family Salicaceae and has had long-lasting effects on those genes with important biological functions., (© 2022. The Author(s).)

Within the genus Salix, there are approximately 350 species native primarily to the northern hemisphere and adapted to a wide range of habitats. This diversity can be exploited to mine novel alleles conferring variation important for production as a bioenergy crop, but also to identify evolutionarily important genes, such as those involved in sex determination. To leverage this diversity, we created a mapping population by crossing 6 Salix species (Salix viminalis, Salix suchowensis, Salix integra, Salix koriyanagi, Salix udensis, and Salix alberti) to common male and female Salix purpurea parents. Each family was genotyped via genotyping-by-sequencing and assessed for kinship and population structure as well as the construction of 16 backcross linkage maps to be used as a genetic resource for breeding and selection. Analyses of population structure resolved both the parents and F1 progeny to their respective phylogenetic section and indicated that the S. alberti parent was misidentified and was most likely S.suchowensis. Sex determining regions were identified on Salix chromosome 15 in the female-informative maps for seven of the eight families indicating that these species share a common female heterogametic ZW sex system. The eighth family, S. integra × S. purpurea, was entirely female and had a truncated chromosome 15. Beyond sex determination, the Salix F1 hybrid common parent population (Salix F1 HCP) introduced here will be useful in characterizing genetic factors underlying complex traits, aid in marker-assisted selection, and support genome assemblies for this promising bioenergy crop., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America.)

Elucidating the genetic factors underlying long-term biological processes remains challenging since the relevant genes and their effects may vary across different developmental stages. In this study, we carried out a large-scale field trial of the progeny of an F1 full-sib pedigree of Salix suchowensis and measured plant height and ground diameter periodically over a time course of 240 days. With the obtained data, we characterized plant growth rhythms and performed time-sequential analyses of quantitative trait loci underlying the dynamic growth of the plants. The dynamic mapping of quantitative trait loci revealed that stem height and ground diameter were under the control of four quantitative trait loci, and the effects of these quantitative trait loci varied greatly throughout the growth process, in which two quantitative trait loci were found to exert a pleiotropic effect determining the correlation between stem height and ground diameter. The analysis of candidate genes in the target genetic intervals showed that the pleiotropic effect of the two quantitative trait loci arises from the colocalization of genes with independent effects on stem height and ground diameter. Further examination of the expression patterns of the candidate genes indicated that height and circumference growth involve different activities of leaf and cambium tissues. This study provides unprecedented information to help us understand the dynamic growth of plants and presents an applicable strategy for elucidating the genetic mechanism underlying a long-term biological process by using plant growth as an example., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Shrub willows ( Salix spp.) are emerging as a viable lignocellulosic, second-generation bioenergy crop with many growth characteristics favorable for marginal lands in New York State and surrounding areas. Willow rust, caused by members of the genus Melampsora , is the most limiting disease of shrub willow in this region and remains extremely understudied. In this study, genetic diversity, genetic structure, and pathogen clonality were examined in Melampsora americana over two growing seasons via genotyping-by-sequencing to identify single-nucleotide polymorphism markers. In conjunction with this project, a reference genome of rust isolate R15-033-03 was generated to aid in variant discovery. Sampling between years allowed regional and site-specific investigation into population dynamics, in the context of both wild and cultivated hosts within high-density plantings. This work revealed that this pathogen is largely panmictic over the sampled areas, with few sites showing moderate genetic differentiation. These data support the hypothesis of sexual recombination between growing seasons because no genotype persisted across the two years of sampling. Additionally, clonality was determined as a driver of pathogen populations within cultivated fields and single shrubs; however, there is also evidence of high genetic diversity of rust isolates in all settings. This work provides a framework for M. americana population structure in the Great Lakes region, providing crucial information that can aid in future resistance breeding efforts.

Background and Aims: The largest genus of Salicaceae sensu lato, Salix, has been shown to consist of two main clades: clade Salix, in which species have XY sex-determination systems (SDSs) on chromosome 7, and clade Vetrix including species with ZW SDSs on chromosome 15. Here, we test the utility of whole genome re-sequencing (WGR) for phylogenomic reconstructions of willows to infer changes between different SDSs., Methods: We used more than 1 TB of WGR data from 70 Salix taxa to ascertain single nucleotide polymorphisms on the autosomes, the sex-linked regions (SLRs) and the chloroplast genomes, for phylogenetic and species tree analyses. To avoid bias, we chose reference genomes from both groups, Salix dunnii from clade Salix and S. purpurea from clade Vetrix., Key Results: Two main largely congruent groups were recovered: the paraphyletic Salix grade and the Vetrix clade. The autosome dataset trees resolved four subclades (C1-C4) in Vetrix. C1 and C2 comprise species from the Hengduan Mountains and adjacent areas and from Eurasia, respectively. Section Longifoliae (C3) grouped within the Vetrix clade but fell into the Salix clade in trees based on the chloroplast dataset analysis. Salix triandra from Eurasia (C4) was revealed as sister to the remaining species of clade Vetrix. In Salix, the polyploid group C5 is paraphyletic to clade Vetrix and subclade C6 is consistent with Argus's subgenus Protitea. Chloroplast datasets separated both Vetrix and Salix as monophyletic, and yielded C5 embedded within Salix. Using only diploid species, both the SLR and autosomal datasets yielded trees with Vetrix and Salix as well-supported clades., Conclusion: WGR data are useful for phylogenomic analyses of willows. The different SDSs may contribute to the isolation of the two major groups, but the reproductive barrier between them needs to be studied., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Salix matsudana (Koidz.) is a widely planted ornamental allotetraploid tree species. Genetic engineering can be used to enhance the tolerance of this species to soil salinization, endowing varieties with the ability to grow along coastlines, thereby mitigating afforestation and protecting the environment. The AP2/ERF family of transcription factors (TFs) plays multidimensional roles in plant biotic/abiotic stress tolerance and plant development. In this study, we cloned the SmAP2-17 gene and performed functional analysis of its role in salt tolerance. This study aims to identify key genes for future breeding of stress-resistant varieties of Salix matsudana., Results: SmAP2-17 was predicted to be a homolog of AP2-like ethylene-responsive transcription factor ANT isoform X2 from Arabidopsis, with a predicted ORF of 2058 bp encoding an estimated protein of 685 amino acids containing two conserved AP2 domains (PF00847.20). SmAP2-17 had a constitutive expression pattern and was localized to the nucleus. The overexpression of the native SmAP2-17 CDS sequence in Arabidopsis did not increase salt tolerance because of the reduced expression level of ectopic SmAP2-17, potentially caused by salt-induced RNAi. Transgenic lines with high expression of optimized SmAP2-17 CDS under salt stress showed enhanced tolerance to salt. Moreover, the expression of general stress marker genes and important salt stress signaling genes, including RD29A, ABI5, SOS3, AtHKT1, and RBohF, were upregulated in SmAP2-17-overexpressed lines, with expression levels consistent with that of SmAP2-17 or optimized SmAP2-17. Promoter activity analysis using dual luciferase analysis showed that SmAP2-17 could bind the promoters of SOS3 and ABI5 to activate their expression, which plays a key role in regulating salt tolerance., Conclusions: The SmAP2-17 gene isolated from Salix matsudana (Koidz.) is a positive regulator that improves the resistance of transgenic plants to salt stress by upregulating SOS3 and ABI5 genes. This study provides a potential functional gene resource for future generation of salt-resistant Salix lines by genetic engineering., (© 2022. The Author(s).)

Many studies have highlighted the complex and diverse basis for heterosis in inbred crops. Despite the lack of a consensus model, it is vital that we turn our attention to understanding heterosis in undomesticated, heterozygous, and polyploid species, such as willow (Salix spp.). Shrub willow is a dedicated energy crop bred to be fast-growing and high yielding on marginal land without competing with food crops. A trend in willow breeding is the consistent pattern of heterosis in triploids produced from crosses between diploid and tetraploid species. Here, we test whether differentially expressed genes are associated with heterosis in triploid families derived from diploid Salix purpurea, diploid Salix viminalis, and tetraploid Salix miyabeana parents. Three biological replicates of shoot tips from all family progeny and parents were collected after 12 weeks in the greenhouse and RNA extracted for RNA-Seq analysis. This study provides evidence that nonadditive patterns of gene expression are correlated with nonadditive phenotypic expression in interspecific triploid hybrids of willow. Expression-level dominance was most correlated with heterosis for biomass yield traits and was highly enriched for processes involved in starch and sucrose metabolism. In addition, there was a global dosage effect of parent alleles in triploid hybrids, with expression proportional to copy number variation. Importantly, differentially expressed genes between family parents were most predictive of heterosis for both field and greenhouse collected traits. Altogether, these data will be used to progress models of heterosis to complement the growing genomic resources available for the improvement of heterozygous perennial bioenergy crops., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

Premise: Gene flow in riparian ecosystems is influenced by landscape features such as orography, climate, and salinity. The downstream increase in genetic diversity (DIGD) hypothesis states that the unidirectionality of the watercourse causes an accumulation of genetic diversity toward downstream populations, while upstream populations are more structured and less diverse, especially in water-dispersed organisms., Methods: We used chloroplast and nuclear microsatellites to characterize genetic diversity, structure, and gene flow patterns among populations of Salix humboldtiana across an elevation and salinity gradient on three rivers (Actopan, Antigua, and Blanco) in Mexico. We used optimization of resistance surface methods to determine whether genetic distances between populations are correlated with landscape features., Results: Positive F IS values evidenced biparental inbreeding in some populations, particularly at higher elevations where lower niche availability constrains colonization and persistence. Four genetic groups were distinguished, corresponding to populations on the Actopan and Antigua rivers and upstream and downstream on the Blanco, but with high admixture between populations on the Actopan and Antigua rivers. Higher gene flow rates were found among proximate populations on the same river than among different rivers. Genetic diversity increased toward the river mouths, in support of the DIGD hypothesis, probably due to greater niche availability and larger population size. Differences among rivers in precipitation patterns and salinity, as well as geographic distance, were significant predictors of gene flow., Conclusions: Our results depict that the DIGD and gene flow patterns in S. humboldtiana result from the complex interaction among physiography, climate, river salinity, and life-history traits of the species., (© 2022 Botanical Society of America.)

Background: Submergence threatens plant growth and survival by decreasing or eliminating oxygen supply. Uncovering the complex regulatory network underlying the tolerance of Salix to submergence and identifying the key regulators are important for molecular-assisted breeding of Salix ., Methods: In this study, we screened germplasm resources of arbor willows and discovered both submergence-tolerant and submergence-sensitive varieties. Then, by performing RNA-seq, we compared the differences between the transcriptomes of two varieties, i.e ., the submergence-tolerant variety "Suliu 795" and the submergence-sensitive variety "Yanliu No. 1," and the different submergence treatment time points to identify the potential mechanisms of submergence in Salix and the unique approaches by which the variety "Suliu 795" possessed a higher tolerance compared to "Yanliu No. 1"., Results: A total of 22,790 differentially expressed genes were identified from 25 comparisons. Using gene ontology annotation and pathway enrichment analysis, the expression pattern of transcriptional factors, important players in hormone signaling, carbohydrate metabolism, and the anaerobic respiration pathway were found to differ significantly between the two varieties. The principal component analysis and qRT-PCR results verified the reliability of the RNA sequencing data. The results of further analysis indicated that "Suliu 795" had higher submergence tolerant activity than "Yanliu No. 1" because of three characteristics: (1) high sensitivity to the probable low oxygen stress and initiation of appropriate responding mechanisms in advance; (2) maintenance of energy homeostasis to prevent energy depletion under hypoxic stress; and (3) keep "quiescence" through fine-tuning the equilibrium between phytohormones GA, SA and ethylene., Competing Interests: The authors declare that they have no competing interests., (© 2022 Chen et al.)

The genetic basis of bird migration has been the focus of several studies. Two willow warbler subspecies (Phylloscopus trochilus trochilus and Phylloscopus trochilus acredula) follow different migratory routes to wintering grounds in Africa. Their breeding populations overlap in contact areas or "migratory divides" located in central Scandinavia and in eastern Poland. Earlier analyses demonstrated that the genetic differences between these two migratory phenotypes are few and cluster on chromosomes 1 and 5. In addition, an amplified fragment length polymorphism-derived biallelic marker (known as WW2) presents steep clines across both migratory divides but failed to be mapped in the genome. Here, we characterize the WW2 marker and describe its two variants (WW2 ancestral and WW2 derived) as portions of long terminal repeat retrotransposons originating from an ancient infection by an endogenous retrovirus. We used quantitative polymerase chain reaction techniques to quantify copy numbers of the WW2 derived variant in the two subspecies and their hybrids. This, together with genome analyses revealed that WW2 derived variants are much more abundant in P. t. acredula and appear embedded in a large repeat-rich region (>12 Mbp), not associated with the divergent regions of chromosomes 1 or 5. However, it might interact with genetic elements controlling migration direction. Testing this hypothesis further will require knowing the exact location of this region, such as by obtaining more complete genome assemblies preferably in combination with techniques like fluorescence in situ hybridization applied to a willow warbler karyotype, and finally to investigate the copy number of this marker in hybrids with known migratory tracks., (© 2021 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Background: Melampsora spp. rusts are the greatest pathogen threat to shrub willow (Salix spp.) bioenergy crops. Genetic resistance is key to limit the effects of these foliar diseases on host response and biomass yield, however, the genetic basis of host resistance has not been characterized. The addition of new genomic resources for Salix provides greater power to investigate the interaction between S. purpurea and M. americana, species commonly found in the Northeast US. Here, we utilize 3' RNA-seq to investigate host-pathogen interactions following controlled inoculations of M. americana on resistant and susceptible F 2 S. purpurea genotypes identified in a recent QTL mapping study. Differential gene expression, network analysis, and eQTL mapping were used to contrast the response to inoculation and to identify associated candidate genes., Results: Controlled inoculation in a replicated greenhouse study identified 19 and 105 differentially expressed genes between resistant and susceptible genotypes at 42 and 66 HPI, respectively. Defense response gene networks were activated in both resistant and susceptible genotypes and enriched for many of the same defense response genes, yet the hub genes of these common response modules showed greater mean expression among the resistant plants. Further, eight and six eQTL hotspots were identified at 42 and 66 HPI, respectively. The combined results of three analyses highlight 124 candidate genes in the host for further analysis while analysis of pathogen RNA showed differential expression of 22 genes, two of which are candidate pathogen effectors., Conclusions: We identified two differentially expressed M. americana transcripts and 124 S. purpurea genes that are good candidates for future studies to confirm their role in conferring resistance., (© 2022. The Author(s).)

Plants acquire multiple resources from the environment and may need to adjust and/or balance their respective resource-use efficiencies to maximize grow and survival, in a locally adaptive manner. In this study, tissue and whole-plant carbon (C) isotopic composition (δ13C) and carbon/nitrogen (C/N) ratios provided long-term measures of use efficiencies for water (WUE) and nitrogen (NUE), and a nitrogen (N) isotopic composition (δ15N)-based mass balance model was used to estimate traits related to N uptake and assimilation in heart-leaved willow (Salix eriocephala Michx.). In an initial common garden experiment consisting of 34 populations, we found population-level variation in δ13C, C/N ratio and δ15N, indicating different patterns in WUE, NUE and N uptake and assimilation. Although there was no relationship between foliar δ13C and C/N ratios among populations, there was a significant negative correlation between these measures across all individuals, implying a genetic and/or plastic trade-off between WUE and NUE not associated with local adaptation. To eliminate any environmental effect, we grew a subset of 21 genotypes hydroponically with nitrate as the sole N source and detected significant variation in δ13C, δ15N and C/N ratios. Variation in δ15N was mainly due to genotypic differences in the nitrate efflux/influx ratio (E/I) at the root. Both experiments suggested clinal variation in δ15N (and thus N uptake efficiency) with latitude of origin, which may relate to water availability and could contribute to global patterns in ecosystem δ15N. There was a tendency for genotypes with higher WUE to come from more water-replete sites with shorter and cooler growing seasons. We found that δ13C, C/N ratio and E/I were not inter-correlated, suggesting that the selection of growth, WUE, NUE and N uptake efficiency can occur without trade-off., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Phellinus igniarius is a medicinal fungus possessing potent therapeutic activity due to the polysaccharides, polyphenols, flavonoids, and other secondary metabolites they contain. Laccases are crucial enzymes involved in lignin degradation in Ph. igniarius and offer great potential to accomplish several bioprocesses. To generate Ph. igniarius strains with high biomass, flavonoid, and laccase activity, we used pulsed light (PL) technology for mutagenesis of Ph. igniarius protoplasts and screened for mutants with high biomass, flavonoid, and laccase activity. At the irradiation power of 100 J, treated distance 8.5 cm, irradiation frequency was 0.5 s/time, three times treatments, after five generations of selection, three mutants were obtained with higher biomass production. Compared with control, the mycelium biomass and the flavonoid production of the screened mutant strain QB72 were increased 20.87% and 53.51%, respectively. The total amount of the accumulated extracellular laccase of the QB72 in the first 6 and 8 days increased 23.38% and 22.37% respectively, and over the total 16 days it increased 9.62%. In addition, RAPD analysis results indicated that the genetic materials of the mutant QB72 were altered. PL mutagenesis method has great potential for developing strains, especially Phellinus.

Long-lived plants face the challenge of ever-increasing mutational burden across their long lifespan. Early sequestration of meristematic stem cells is supposed to efficiently slow down this process, but direct measurement of somatic mutations that accompanies segregated cell lineages in plants is still rare. Here, we tracked somatic mutations in 33 leaves and 22 adventitious roots from 22 stem-cuttings across eight major branches of a shrub willow (Salix suchowensis). We found that most mutations propagated separately in leaves and roots, providing clear evidence for early segregation of underlying cell lineages. By combining lineage tracking with allele frequency analysis, our results revealed a set of mutations shared by distinct branches, but were exclusively present in leaves and not in roots. These mutations were likely propagated by rapidly dividing somatic cell lineages which survive several iterations of branching, distinct from the slowly dividing axillary stem cell lineages. Leaf is thus contributed by both slowly and rapidly dividing cell lineages, leading to varied fixation chances of propagated mutations. By contrast, each root likely arises from a single founder cell within the adventitious stem cell lineages. Our findings give straightforward evidence that early segregation of meristems slows down mutation accumulation in axillary meristems, implying a plant "germline" paralog to the germline of animals through convergent evolution., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Growth responses and cadmium (Cd) accumulation in willow cultivars help determine their potential in remediation of Cd-contaminated conditions. Seventeen willow cultivars, including hybrids and their parents, were grown in hydroponic conditions in a greenhouse, and their capacity for Cd tolerance and accumulation was compared. The results showed that shoot length, biomass production, and concentrations of photosynthetic pigments were significantly affected by 10 μM and 30 μM Cd treatments compared with the control. Biomass production varied across all cultivars and treatments, with maximum shoot dry weight in Owasco grown in 10 μM Cd (11.7 ± 4.5 g plant -1 ), and minimum in FC187 in 30 μM Cd (0.3 ± 0.1 g plant -1 ). Furthermore, shoot growth proved to be more sensitive to Cd than root growth. Cultivars tolerance to Cd stress varied as indicated by tolerance indices (TIs) ranging from 0.13 to 1.81 for shoots, and from 0.49 to 2.63 for roots. Cd accumulation also differed across treatments and cultivars, with average concentration of 217.49 μg. g -1 in shoots and 478.47 μg. g -1 in roots. Total amounts of Cd in all plant parts ranged from 38.98 to 4116.05 μg per plant, and cultivars SX64, Cicero, and Owasco exhibited a translocation factor (TF) of greater than 1. The correlation and path coefficient analyses demonstrated that shoot biomass reflected Cd transport and phytoextraction ability in selected willow cultivars. Our results also revealed that cultivars with higher leaf Cd concentration could be used as Cd-efficient parents to generate hybrids for Cd phytoextraction. Cultivars with higher biomass production translocated and accumulated more Cd in their aerial parts, and this finding will facilitate yield-based selection of candidates for Cd phytoextraction and for bioenergy production., (Copyright © 2021 Elsevier Ltd. All rights reserved.)