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The genome of Populus davidiana, a keystone aspen species, has been sequenced to improve our understanding of the evolutionary and functional genomics of the Populus genus. The Hi-C scaffolding genome assembly resulted in a 408.1 Mb genome with 19 pseudochromosomes. The BUSCO assessment revealed that 98.3% of the genome matched the embryophytes dataset. A total of 31,862 protein-coding sequences were predicted, of which 31,619 were functionally annotated. The assembled genome was composed of 44.9% transposable elements. These findings provide new knowledge about the characteristics of the P. davidiana genome and will facilitate comparative genomics and evolutionary research on the genus Populus., (© 2023. The Author(s).)

Both anthocyanins and lignins are essential secondary metabolites in plant growth and development. Their biosynthesis is metabolically interconnected and diverges in the central metabolite 4-coumaroyl CoA of the phenylpropanoid pathway. Considerable progress has been made in understanding transcriptional regulation of genes involved in lignin and anthocyanin synthesis pathways, but the concerted regulation of these pathways is not yet fully understood. Here, we functionally characterized PtrMYB120, a R2R3-MYB transcription factor from Populus trichocarpa. Overexpression of PtrMYB120 in a hybrid poplar (i.e., 35S::PtrMYB120) was associated with increased anthocyanin (i.e., cyanidin 3-O-glucoside) accumulation and upregulation of anthocyanin biosynthetic genes. However, transgenic poplars with dominant suppression of PtrMYB120 function achieved by fusing the ERF-associated amphiphilic repression motif to PtrMYB120 (i.e., 35S::PtrMYB120-SRDX) had a dramatic decrease in not only anthocyanin but also Klason lignin content with downregulation of both anthocyanin and lignin biosynthetic genes. Indeed, 35S::PtrMYB120-SRDX poplars had irregularly shaped xylem vessels with reduced S-lignin content in stems, which was proportionally related to the level of the introduced PtrMYB120-SRDX gene. Furthermore, protoplast-based transcriptional activation assay using the PtrMYB120-GR system suggested that PtrMYB120 directly regulates genes involved in both anthocyanin and lignin biosynthesis, including chalcone synthase and ferulate-5 hydroxylase. Interestingly, the saccharification efficiency of line #6 of 35S::PtrMYB120-SRDX poplars, which had slightly reduced lignin content with a normal growth phenotype, was dramatically enhanced (>45%) by NaOH treatment. Taken together, our results suggest that PtrMYB120 functions as a positive regulator of both anthocyanin and lignin biosynthetic pathways and can be targeted to enhance saccharification efficiency in woody perennials., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The CRISPR/Cas9 system has been used for genome editing in several plant species; however, there are few reports on its use in trees. Here, CRISPR/Cas9 was used to mutate a target gene in Populus alba × Populus glandulosa hybrid poplars. The hybrid poplar is routinely used in molecular biological studies due to the well-established Agrobacterium-mediated transformation method. A single guide RNA (sgRNA) with reported high mutation efficiency in other popular species was designed with a protospacer adjacent motif sequence for the phytoene desaturase 1 (PagPDS1) gene. The pHSE/Cas9-PagPDS1 sgRNA vector was delivered into hybrid poplar cells using Agrobacterium-mediated transformation. The transgenic plants were propagated and classified them into three groups according to their phenotypes. Among a total of 110 lines of transgenic hybrid poplars, 82 lines showed either an albino or a pale green phenotype, indicating around 74.5% phenotypic mutation efficiency of the PagPDS1 gene. The albino phenotypes were observed when the CRISPR/Cas9-mediated mutations in both PagPDS1 alleles in the transgenic plants. There was no off-target modification of the PagPDS2 gene, which has a potential sgRNA target sequence with two mismatches. The results confirmed that the sgRNA can specifically edit PagPDS1 rather than PagPDS2, indicating that CRISPR/Cas9-mediated genome editing can effectively induce target mutations in the hybrid poplar. This technique will be useful to improve tree quality in hybrid poplars (P. alba × P. glandulosa); for example, by enhancing biomass or stress tolerance., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Caffeoyl shikimate esterase (CSE) has been shown to play an important role in lignin biosynthesis in plants and is, therefore, a promising target for generating improved lignocellulosic biomass crops for sustainable biofuel production. Populus spp. has two CSE genes ( CSE1 and CSE2 ) and, thus, the hybrid poplar ( Populus alba × P. glandulosa ) investigated in this study has four CSE genes. Here, we present transgenic hybrid poplars with knockouts of each CSE gene achieved by CRISPR/Cas9. To knockout the CSE genes of the hybrid poplar, we designed three single guide RNAs (sg1-sg3), and produced three different transgenic poplars with either CSE1 (CSE1-sg2), CSE2 (CSE2-sg3), or both genes (CSE1/2-sg1) mutated. CSE1-sg2 and CSE2-sg3 poplars showed up to 29.1% reduction in lignin deposition with irregularly shaped xylem vessels. However, CSE1-sg2 and CSE2-sg3 poplars were morphologically indistinguishable from WT and showed no significant differences in growth in a long-term living modified organism (LMO) field-test covering four seasons. Gene expression analysis revealed that many lignin biosynthetic genes were downregulated in CSE1-sg2 and CSE2-sg3 poplars. Indeed, the CSE1-sg2 and CSE2-sg3 poplars had up to 25% higher saccharification efficiency than the WT control. Our results demonstrate that precise editing of CSE by CRISPR/Cas9 technology can improve lignocellulosic biomass without a growth penalty.

Drought stress is one of the major environmental problems in the growth of crops and woody perennials, but it is getting worse due to the global climate crisis. XERICO, a RING (Really Interesting New Gene) zinc-finger E3 ubiquitin ligase, has been shown to be a positive regulator of drought tolerance in plants through the control of abscisic acid (ABA) homeostasis. We characterized a poplar ( Populus trichocarpa ) RING protein family and identified the closest homolog of XERICO called PtXERICO . Expression of PtXERICO is induced by both salt and drought stress, and by ABA treatment in poplars. Overexpression of PtXERICO in Arabidopsis confers salt and ABA hypersensitivity in young seedlings, and enhances drought tolerance by decreasing transpirational water loss. Consistently, transgenic hybrid poplars overexpressing PtXERICO demonstrate enhanced drought tolerance with reduced transpirational water loss and ion leakage. Subsequent upregulation of genes involved in the ABA homeostasis and drought response was confirmed in both transgenic Arabidopsis and poplars. Taken together, our results suggest that PtXERICO will serve as a focal point to improve drought tolerance of woody perennials.

Patatin-liked phospholipase A (pPLAs) are major lipid acyl hydrolases that participate in various biological functions in plant growth and development. Previously, a ginseng-derived pPLAIII homolog was reported to reduce lignin content in Arabidopsis. This led us to evaluate its possible usefulness as a biomass source in wood plant. Herein, we report that there are six members in the pPLAIII gene family in poplar. Overexpression of pPLAIIIβ derived from ginseng resulted in a reduced plant height with radially expanded stem growth in hybrid poplars. Compared with the wild type (WT), the chlorophyll content was increased in the overexpression poplar lines, whereas the leaf size was smaller. The secondary cell wall structure in overexpression lines was also altered, exhibiting reduced lignification in the xylem. Two transcription factors, MYB92 and MYB152, which control lignin biosynthesis, were downregulated in the overexpression lines. The middle xylem of the overexpression line showed heavy thickening, making it thicker than the other xylem parts and the WT xylem, which rather could have been contributed by the presence of more cellulose in the selected surface area. Taken together, the results suggest that PgpPLAIIIβ plays a role not only in cell elongation patterns, but also in determining the secondary cell wall composition., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Drought and salt stresses are major environmental constraints on forest productivity. To identify genes responsible for stress tolerance, we conducted a genome-wide analysis in poplar (Populus alba × Populus glandulosa) leaves exposed to drought and salt (NaCl) stresses. We investigated gene expression at the mRNA level using oligonucleotide microarrays containing 44,718 genes from Populus trichocarpa. A total of 1604 and 1042 genes were up-regulated (≥2-fold; P value < 0.05) by drought and salt stresses, respectively, and 765 genes were up-regulated by both stresses. In addition, 2742 and 1685 genes were down-regulated by drought and salt stresses, respectively, and 1564 genes were down-regulated by both stresses. The large number of genes regulated by both stresses suggests that crosstalk occurs between the drought and salt stress responses. Most up-regulated genes were involved in functions such as subcellular localization, signal transduction, metabolism, and transcription. Among the up-regulated genes, we identified 47 signaling proteins, 65 transcription factors, and 43 abiotic stress-related genes. Several genes were modulated by only one of the two stresses. About 25% of the genes significantly regulated by these stresses are of unknown function, suggesting that poplar may provide an opportunity to discover novel stress-related genes., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

The gene expression profiles of hybrid poplar (Populus alba × Populus tremula var. glandulosa) cells in suspension culture after exposure to salinity (NaCl) induced stress were examined by constructing two suppression subtractive hybridization (SSH) libraries. cDNA from non-treated cells was used as a driver and cDNA samples from cell suspension cultures exposed to 150 mM NaCl for 2 or 10 h were used as testers. Randomly selected clones from each SSH library were sequenced and 727 high-quality expressed sequence tags (ESTs) were obtained and analyzed. Four novel ESTs were identified. Between the two libraries, 542 unique SSH clones were selected for placement on a cDNA microarray. In total, 18 differentially expressed genes were identified with 4 and 12 genes being significantly differentially expressed 2 and 10 h after the treatment, respectively. Genes related to metabolism and protein synthesis and several genes whose protein products are implicated in salt or other abiotic stress-related responses were expressed in the salt-stressed cells., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Water uptake across cell membranes is a principal requirement for plant growth at both the cellular and whole-plant levels; water movement through plant membranes is regulated by aquaporins (AQPs) or major intrinsic proteins (MIPs). We examined the expression characteristics of the poplar plasma membrane intrinsic protein 1 gene (PatPIP1), a type of MIP, which was isolated from a suspension cell cDNA library of Populus alba×P. tremula var. glandulosa. Examination of protoplasts expressing the p35S-PatPIP1::sGFP fusion protein revealed that the protein was localized in the plasma membrane. Northern blot analysis revealed that the gene was strongly expressed in poplar roots and leaves. Gene expression was inducible by abiotic factors including drought, salinity, cold temperatures and wounding, and also by plant hormones including gibberellic acid, jasmonic acid and salicylic acid. Since we found that the PatPIP1 gene was strongly expressed in response to mannitol, NaCl, jasmonic acid and wounding, we propose that PatPIP1 plays an essential role in the defense of plants against water stress., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Osmotic stress induces changes in the expression of various genes including those associated with drought tolerance, cell wall metabolism and defense. We isolated 852 cDNA clones, the expression of which is induced by osmotic stress, from cells of a hybrid poplar (Populus alba x Populus tremula var. glandulosa) by suppression subtractive hybridization after mannitol treatment. We examined how stress affected their expression using cDNA microarray analysis, which identified 104 genes significantly up-regulated by osmotic stress. These include genes with functions related to transcription, signal transduction, cell wall metabolism and defense. Other gene transcripts encoding cysteine protease and aquaporin are also up-regulated during osmotic stress. The function of about one-third of the genes in poplar cells that were significantly up-regulated by stress is not known, suggesting that the cell suspension may offer an opportunity of finding novel genes otherwise never expressed and that we still need more information at the molecular level., (Copyright 2009 Elsevier Masson SAS. All rights reserved.)

Dehydrins are group II, late embryogenesis abundant proteins that act putatively as chaperones in stressed plants. To elucidate the function of dehydrins in poplar, we isolated the SK(2)-type dehydrin gene Podhn from Populus alba x P. tremula var. glandulosa suspension cells and analyzed its expression following treatments of abiotic stress, wounding and plant growth regulator. Sequence homology and phylogenetic analyses indicate Podhn encodes an acidic dehydrin (pI 5.14, 277 amino acids, predicted size 25.6 kDa) containing two lysine-rich "K-segments" and a 7-serine residue "S-segment", both characteristic of SK(2)-type dehydrins. Southern blots show Podhn genes form a small gene family in poplar. Podhn was expressed in all tissues examined under unstressed conditions, but most strongly in cell suspensions (especially in the stationary phase). Drought, salt, cold and exogenous abscisic acid (ABA) treatments enhanced Podhn expression, while wounding and jasmonic acid caused its reduction. Therefore, Podhn might be involved in ABA or stress response.

Complex sequences of morphological and biochemical changes occur during the developmental course of a batch plant cell culture. However, little information is available about the changes in gene expression that could explain these changes, because of the difficulties involved in isolating specific cellular events or developmental phases in the overlapping phases of cell growth. In an attempt to obtain such information we have examined the global growth phase-dependent gene expression of poplar cells in suspension cultures by cDNA microarray analysis. Our results reveal that significant changes occur in the expression of genes with functions related to protein synthesis, cell cycling, hormonal responses and cell wall biosynthesis, as cultures progress from initiation to senescence, that are highly correlated with observed developmental and physiological changes in the cells. Genes encoding protein kinases, calmodulin and proteins involved in both ascorbate metabolism and water-limited stress responses also showed strong stage-specific expression patterns. Our report provides fundamental information on molecular mechanisms that control cellular changes throughout the developmental course of poplar cell cultures.

To elucidate the precise functions of peroxidase in poplar (Populus alba x P. tremula var. glandulosa), we cloned a peroxidase gene (PoPOD1) from poplar suspension culture cells and examined its expression pattern in response to various stresses. PoPOD1 showed the highest homology with a bacterial-induced peroxidase gene from cotton (Gossypium hirsutum L.). The PoPOD1 gene encodes a putative 316 amino acid protein with an N-terminal signal peptide of 23 residues. The DNA blot analysis indicated that PoPOD1 is a single copy gene in the poplar genome. The RNA blot analyses indicated that PoPOD1 shows cell-culture-specific expression. Expression of PoPOD1 is down-regulated by various treatments including treatment with some metals, NaCl, methyl viologen and polyethylene glycol, and by the plant growth regulators, jasmonic acid (JA) and gibberellic acid (GA(3)). The gene is significantly up-regulated by the bacterial-elicitor laminarin and by wounding. Thus, PoPOD1 gene expression is sensitively and specifically regulated at the transcription level. Because both JA and GA3 appear to be involved in the regulation of PoPOD1 expression in poplar cells, we postulate that the peroxidase encoded by PoPOD1 plays a pivotal role in defense against pathogen invasion, possibly through the formation of a cell wall barrier over the wound.

Copper chaperone (CCH) is upregulated during Arabidopsis (Arabidopsis thaliana L. Columbia) leaf senescence, suggesting that it mobilizes certain metal ions in leaves and transports them to other growing parts of the plants. The CCHs are also involved in defense mechanisms against oxidative stress in Arabidopsis and tomato (Lycopersicon esculentum Mill. cv. 'Ailsa Craig'). To elucidate the functions of CCH in poplar, we cloned a CCH gene (PoCCH) from Populus (Populus alba x P. tremula var. glandulosa) suspension cells and tested its expression in response to various treatments including heavy metals, plant growth regulators and abiotic stresses. The PoCCH cDNA is 540 bp in length, including a 55-bp 5' noncoding domain, a 258-bp open reading frame (ORF), and 227-bp 3' termination region. The coding region of PoCCH represents a putative 85-amino-acid protein with a molecular weight of 8.9 kDa. The deduced amino acid sequence of the PoCCH gene product is 87 and 78% identical to those of tomato and Arabidopsis, respectively, with a high degree of conservation in both the metal-binding and lysine-rich regions. However, the PoCCH gene product lacks the C-terminal extension identified in Arabidopsis CCH. Southern blot analysis suggested that the PoCCH gene is present in low copy numbers in poplar. The expression of PoCCH increased under copper deprivation conditions. The expression of PoCCH was down-regulated by high concentrations of copper, whereas some metals, such as aluminum and zinc, markedly induced PoCCH, and others including cadmium, cobalt and lead had little effect on PoCCH expression. The plant growth regulator jasmonic acid caused an increase in PoCCH transcript whereas abscisic acid, salicylic acid and gibberellic acid did not. The gene was highly induced when cells were exposed to physical stress by high-speed agitation on a gyratory shaker. Other effective inducers of PoCCH expression in suspension culture were methyl viologen and NaCl. Thus, PoCCH does not respond to all stresses, but responds specifically to certain metals and abiotic stresses that induce oxidative damage. Our results suggest that JA is involved in regulating PoCCH expression in poplar cells.