Multiplex CRISPR editing of wood for sustainable fiber production.

The domestication of forest trees for a more sustainable fiber bioeconomy has long been hindered by the complexity and plasticity of lignin, a biopolymer in wood that is recalcitrant to chemical and enzymatic degradation. Here, we show that multiplex CRISPR editing enables precise woody feedstock design for combinatorial improvement of lignin composition and wood properties. By assessing every possible combination of 69,123 multigenic editing strategies for 21 lignin biosynthesis genes, we deduced seven different genome editing strategies targeting the concurrent alteration of up to six genes and produced 174 edited poplar variants. CRISPR editing increased the wood carbohydrate-to-lignin ratio up to 228% that of wild type, leading to more-efficient fiber pulping. The edited wood alleviates a major fiber-production bottleneck regardless of changes in tree growth rate and could bring unprecedented operational efficiencies, bioeconomic opportunities, and environmental benefits.

Overexpression of a gibberellin 20-oxidase gene in poplar xylem led to an increase in the size of nanocellulose fibrils and improved paper properties.

Cellulose, the major component of secondary cell walls, is the most abundant renewable long-chain polymer on earth. Nanocellulose has become a prominent nano-reinforcement agent for polymer matrices in various industries. We report the generation of transgenic hybrid poplar overexpressing the Arabidopsis gibberellin 20-oxidase1 gene driven by a xylem-specific promoter to increase gibberellin (GA) biosynthesis in wood. X-ray diffraction (XRD) and sum frequency generation spectroscopic (SFG) analyses showed that cellulose in transgenic trees was less crystalline, but the crystal size was larger. The nanocellulose fibrils prepared from transgenic wood had an increased size compared to those from wild type. When such fibrils were used as a reinforcing agent in sheet paper preparation, the mechanical strength of the paper was significantly enhanced. Engineering the GA pathway can therefore affect nanocellulose properties, providing a new strategy for expanding nanocellulose applications.

Chromosome-level genome assembly of a triploid poplar Populus alba 'Berolinensis'.

Many recent studies have provided significant insights into polyploid breeding, but limited research has been carried out on trees. The genomic information needed to understand growth and response to abiotic stress in polyploidy trees is largely unknown, but has become critical due to the threats to forests imposed by climate change. Populus alba 'Berolinensis,' also known "Yinzhong poplar," is a triploid poplar from northeast China. This hybrid triploid poplar is widely used as a landscape ornamental and in urban forestry owing to its adaptation to adverse environments and faster growth than its parental diploid. It is an artificially synthesized male allotriploid hybrid, with three haploid genomes of P. alba 'Berolinensis' originating from different poplar species, so it is attractive for studying polyploidy genomic mechanisms in heterosis. In this study, we focused on the allelic genomic interactions in P. alba 'Berolinensis,' and generated a high-quality chromosome-level genome assembly consisting of 19 allelic chromosomes. Its three haploid chromosome sets are polymorphic with an average of 25.73 nucleotide polymorphism sites per kilobase. We found that some stress-related genes such as RD22 and LEA7 exhibited sequence differences between different haploid genomes. The genome assembly has been deposited in our polyploid genome online analysis website TreeGenomes (https://www.treegenomes.com). These polyploid genome-related resources will provide a critical foundation for the molecular breeding of P. alba 'Berolinensis' and help us uncover the allopolyploidization effects of heterosis and abiotic stress resistance and traits of polyploidy species in the future.

The double flower variant of yellowhorn is due to a LINE1 transposon-mediated insertion.

As essential organs of reproduction in angiosperms, flowers, and the genetic mechanisms of their development have been well characterized in many plant species but not in the woody tree yellowhorn (Xanthoceras sorbifolium). Here, we focused on the double flower phenotype in yellowhorn, which has high ornamental value. We found a candidate C-class gene, AGAMOUS1 (XsAG1), through bovine serum albumin sequencing and genetics analysis with a Long Interpersed Nuclear Elements 1 (LINE1) transposable element fragment (Xsag1-LINE1-1) inserted into its second intron that caused a loss-of-C-function and therefore the double flower phenotype. In situ hybridization of XsAG1 and analysis of the expression levels of other ABC genes were used to identify differences between single- and double-flower development processes. These findings enrich our understanding of double flower formation in yellowhorn and provide evidence that transposon insertions into genes can reshape plant traits in forest trees.

Ubiquitinated DA1 negatively regulates vascular cambium activity through modulating the stability of WOX4 in Populus.

Activity of the vascular cambium gives rise to secondary xylem for wood formation in trees. The transcription factor WUSCHEL-related HOMEOBOX4 (WOX4) is a central regulator downstream of the hormone and peptide signaling pathways that maintain cambial activity. However, the genetic regulatory network underlying WOX4-mediated wood formation at the post-transcriptional level remains to be elucidated. In this study, we identified the ubiquitin receptor PagDA1 in hybrid poplar (Populus alba × Populus glandulosa clone 84K) as a negative regulator of wood formation, which restricts cambial activity during secondary growth. Overexpression of PagDA1 in poplar resulted in a relatively reduced xylem due to decreased cambial cell division. By contrast, mutation of PagDA1 by CRISPR/Cas9 resulted in an increased cambial cell activity and promoted xylem formation. Genetic analysis demonstrated that PagDA1 functions antagonistically in a common pathway as PagWOX4 to regulate cambial activity. We propose that PagDA1 physically associates with PagWOX4 and modulates the degradation of PagWOX4 by the 26S proteasome. Moreover, genetic analysis revealed that PagDA1 exerts its negative effect on cambial development by modulating the stability of PagWOX4 in a ubiquitin-dependent manner mediated by the E3 ubiquitin ligase PagDA2. In sum, we have identified a cambial regulatory protein complex, PagDA1-PagWOX4, as a potential target for wood biomass improvement.

Gene sdaB Is Involved in the Nematocidal Activity of Enterobacter ludwigii AA4 Against the Pine Wood Nematode Bursaphelenchus xylophilus.

Bursaphelenchus xylophilus, a plant parasitic nematode, is the causal agent of pine wilt, a devastating forest tree disease. Essentially, no efficient methods for controlling B. xylophilus and pine wilt disease have yet been developed. Enterobacter ludwigii AA4, isolated from the root of maize, has powerful nematocidal activity against B. xylophilus in a new in vitro dye exclusion test. The corrected mortality of the B. xylophilus treated by E. ludwigii AA4 or its cell extract reached 98.3 and 98.6%, respectively. Morphological changes in B. xylophilus treated with a cell extract from strain AA4 suggested that the death of B. xylophilus might be caused by an increased number of vacuoles in non-apoptotic cell death and the damage to tissues of the nematodes. In a greenhouse test, the disease index of the seedlings of Scots pine (Pinus sylvestris) treated with the cells of strain AA4 plus B. xylophilus or those treated by AA4 cell extract plus B. xylophilus was 38.2 and 30.3, respectively, was significantly lower than 92.5 in the control plants treated with distilled water and B. xylophilus. We created a sdaB gene knockout in strain AA4 by deleting the gene that was putatively encoding the beta-subunit of L-serine dehydratase through Red homologous recombination. The nematocidal and disease-suppressing activities of the knockout strain were remarkably impaired. Finally, we revealed a robust colonization of P. sylvestris seedling needles by E. ludwigii AA4, which is supposed to contribute to the disease-controlling efficacy of strain AA4. Therefore, E. ludwigii AA4 has significant potential to serve as an agent for the biological control of pine wilt disease caused by B. xylophilus.

The flying spider-monkey tree fern genome provides insights into fern evolution and arborescence.

To date, little is known about the evolution of fern genomes, with only two small genomes published from the heterosporous Salviniales. Here we assembled the genome of Alsophila spinulosa, known as the flying spider-monkey tree fern, onto 69 pseudochromosomes. The remarkable preservation of synteny, despite resulting from an ancient whole-genome duplication over 100 million years ago, is unprecedented in plants and probably speaks to the uniqueness of tree ferns. Our detailed investigations into stem anatomy and lignin biosynthesis shed new light on the evolution of stem formation in tree ferns. We identified a phenolic compound, alsophilin, that is abundant in xylem, and we provided the molecular basis for its biosynthesis. Finally, analysis of demographic history revealed two genetic bottlenecks, resulting in rapid demographic declines of A. spinulosa. The A. spinulosa genome fills a crucial gap in the plant genomic landscape and helps elucidate many unique aspects of tree fern biology.

Enzyme Complexes of Ptr4CL and PtrHCT Modulate Co-enzyme A Ligation of Hydroxycinnamic Acids for Monolignol Biosynthesis in Populus trichocarpa.

Co-enzyme A (CoA) ligation of hydroxycinnamic acids by 4-coumaric acid:CoA ligase (4CL) is a critical step in the biosynthesis of monolignols. Perturbation of 4CL activity significantly impacts the lignin content of diverse plant species. In Populus trichocarpa, two well-studied xylem-specific Ptr4CLs (Ptr4CL3 and Ptr4CL5) catalyze the CoA ligation of 4-coumaric acid to 4-coumaroyl-CoA and caffeic acid to caffeoyl-CoA. Subsequently, two 4-hydroxycinnamoyl-CoA:shikimic acid hydroxycinnamoyl transferases (PtrHCT1 and PtrHCT6) mediate the conversion of 4-coumaroyl-CoA to caffeoyl-CoA. Here, we show that the CoA ligation of 4-coumaric and caffeic acids is modulated by Ptr4CL/PtrHCT protein complexes. Downregulation of PtrHCTs reduced Ptr4CL activities in the stem-differentiating xylem (SDX) of transgenic P. trichocarpa. The Ptr4CL/PtrHCT interactions were then validated in vivo using biomolecular fluorescence complementation (BiFC) and protein pull-down assays in P. trichocarpa SDX extracts. Enzyme activity assays using recombinant proteins of Ptr4CL and PtrHCT showed elevated CoA ligation activity for Ptr4CL when supplemented with PtrHCT. Numerical analyses based on an evolutionary computation of the CoA ligation activity estimated the stoichiometry of the protein complex to consist of one Ptr4CL and two PtrHCTs, which was experimentally confirmed by chemical cross-linking using SDX plant protein extracts and recombinant proteins. Based on these results, we propose that Ptr4CL/PtrHCT complexes modulate the metabolic flux of CoA ligation for monolignol biosynthesis during wood formation in P. trichocarpa.

Corrigendum: BEL1-like Homeodomain Protein BLH6a Is a Negative Regulator of CAld5H2 in Sinapyl Alcohol Monolignol Biosynthesis in Poplar.

[This corrects the article DOI: 10.3389/fpls.2021.695223.].

Single-cell RNA sequencing reveals a high-resolution cell atlas of xylem in Populus.

High-throughput single-cell RNA sequencing (scRNA-seq) has advantages over traditional RNA-seq to explore spatiotemporal information on gene dynamic expressions in heterogenous tissues. We performed Drop-seq, a method for the dropwise sequestration of single cells for sequencing, on protoplasts from the differentiating xylem of Populus alba × Populus glandulosa. The scRNA-seq profiled 9,798 cells, which were grouped into 12 clusters. Through characterization of differentially expressed genes in each cluster and RNA in situ hybridizations, we identified vessel cells, fiber cells, ray parenchyma cells and xylem precursor cells. Diffusion pseudotime analyses revealed the differentiating trajectory of vessels, fiber cells and ray parenchyma cells and indicated a different differentiation process between vessels and fiber cells, and a similar differentiation process between fiber cells and ray parenchyma cells. We identified marker genes for each cell type (cluster) and key candidate regulators during developmental stages of xylem cell differentiation. Our study generates a high-resolution expression atlas of wood formation at the single cell level and provides valuable information on wood formation.

BEL1-like Homeodomain Protein BLH6a Is a Negative Regulator of CAl5H2 in Sinapyl Alcohol Monolignol Biosynthesis in Poplar.

Lignin is one of the major components of xylem cell walls in tree stems. The lignin in the wood of most flowering plants (dicotyledonous angiosperms) is typically polymerized from three monolignol precursors, coniferyl alcohol, sinapyl alcohol, and p-coumaroyl alcohol, resulting in guaiacyl (G), syringyl (S), and hydroxyphenyl (H) subunits, respectively. In this study, we focus on the transcriptional regulation of a coniferaldehyde 5-hydroxylase (CAld5H2) gene, which encodes a key enzyme for sinapyl alcohol biosynthesis. We carried out a yeast one-hybrid (Y1H) screen to identify candidate upstream transcription factors (TFs) regulating CAld5H2. We obtained 12 upstream TFs as potential regulators of CAld5H2. One of these TF genes, BLH6a, encodes a BEL1-like homeodomain (BLH) protein and negatively regulated the CAld5H2 promoter activity. The direct regulation of CAld5H2 promoter by BLH6a was supported by chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) and dominant repression of BLH6a in transgenic plants. Luciferase complementation imaging analyses showed extensive protein-protein interactions among these 12 TFs. We propose that BLH6a is a negative regulator of CAld5H2, which acts through combinatorial regulation of multiple TFs for sinapyl alcohol (S monolignol) biosynthesis in poplar.

Microbial Interactions Within Multiple-Strain Biological Control Agents Impact Soil-Borne Plant Disease.

Major losses of crop yield and quality caused by soil-borne plant diseases have long threatened the ecology and economy of agriculture and forestry. Biological control using beneficial microorganisms has become more popular for management of soil-borne pathogens as an environmentally friendly method for protecting plants. Two major barriers limiting the disease-suppressive functions of biocontrol microbes are inadequate colonization of hosts and inefficient inhibition of soil-borne pathogen growth, due to biotic and abiotic factors acting in complex rhizosphere environments. Use of a consortium of microbial strains with disease inhibitory activity may improve the biocontrol efficacy of the disease-inhibiting microbes. The mechanisms of biological control are not fully understood. In this review, we focus on bacterial and fungal biocontrol agents to summarize the current state of the use of single strain and multi-strain biological control consortia in the management of soil-borne diseases. We discuss potential mechanisms used by microbial components to improve the disease suppressing efficacy. We emphasize the interaction-related factors to be considered when constructing multiple-strain biological control consortia and propose a workflow for assembling them by applying a reductionist synthetic community approach.

Involvement of CesA4, CesA7-A/B and CesA8-A/B in secondary wall formation in Populus trichocarpa wood.

Cellulose synthase A genes (CesAs) are responsible for cellulose biosynthesis in plant cell walls. In this study, functions of secondary wall cellulose synthases PtrCesA4, PtrCesA7-A/B and PtrCesA8-A/B were characterized during wood formation in Populus trichocarpa (Torr. & Gray). CesA RNAi knockdown transgenic plants exhibited stunted growth, narrow leaves, early necrosis, reduced stature, collapsed vessels, thinner fiber cell walls and extended fiber lumen diameters. In the RNAi knockdown transgenics, stems exhibited reduced mechanical strength, with reduced modulus of rupture (MOR) and modulus of elasticity (MOE). The reduced mechanical strength may be due to thinner fiber cell walls. Vessels in the xylem of the transgenics were collapsed, indicating that water transport in xylem may be affected and thus causing early necrosis in leaves. A dramatic decrease in cellulose content was observed in the RNAi knockdown transgenics. Compared with wildtype, the cellulose content was significantly decreased in the PtrCesA4, PtrCesA7 and PtrCesA8 RNAi knockdown transgenics. As a result, lignin and xylem contents were proportionally increased. The wood composition changes were confirmed by solid-state NMR, two-dimensional solution-state NMR and sum-frequency-generation vibration (SFG) analyses. Both solid-state nuclear magnetic resonance (NMR) and SFG analyses demonstrated that knockdown of PtrCesAs did not affect cellulose crystallinity index. Our results provided the evidence for the involvement of PtrCesA4, PtrCesA7-A/B and PtrCesA8-A/B in secondary cell wall formation in wood and demonstrated the pleiotropic effects of their perturbations on wood formation.

Hierarchical Transcription Factor and Chromatin Binding Network for Wood Formation in Black Cottonwood (Populus trichocarpa).

Wood remains the world's most abundant and renewable resource for timber and pulp and is an alternative to fossil fuels. Understanding the molecular regulation of wood formation can advance the engineering of wood for more efficient material and energy productions. We integrated a black cottonwood (Populus trichocarpa) wood-forming cell system with quantitative transcriptomics and chromatin binding assays to construct a transcriptional regulatory network (TRN) directed by a key transcription factor (TF), PtrSND1-B1 (secondary wall-associated NAC-domain protein). The network consists of four layers of TF-target gene interactions with quantitative regulatory effects, describing the specificity of how the regulation is transduced through these interactions to activate cell wall genes (effector genes) for wood formation. PtrSND1-B1 directs 57 TF-DNA interactions through 17 TFs transregulating 27 effector genes. Of the 57 interactions, 55 are novel. We tested 42 of these 57 interactions in 30 genotypes of transgenic P. trichocarpa and verified that ∼90% of the tested interactions function in vivo. The TRN reveals common transregulatory targets for distinct TFs, leading to the discovery of nine TF protein complexes (dimers and trimers) implicated in regulating the biosynthesis of specific types of lignin. Our work suggests that wood formation may involve regulatory homeostasis determined by combinations of TF-DNA and TF-TF (protein-protein) regulations.

CAD1 and CCR2 protein complex formation in monolignol biosynthesis in Populus trichocarpa.

Lignin is the major phenolic polymer in plant secondary cell walls and is polymerized from monomeric subunits, the monolignols. Eleven enzyme families are implicated in monolignol biosynthesis. Here, we studied the functions of members of the cinnamyl alcohol dehydrogenase (CAD) and cinnamoyl-CoA reductase (CCR) families in wood formation in Populus trichocarpa, including the regulatory effects of their transcripts and protein activities on monolignol biosynthesis. Enzyme activity assays from stem-differentiating xylem (SDX) proteins showed that RNAi suppression of PtrCAD1 in P. trichocarpa transgenics caused a reduction in SDX CCR activity. RNAi suppression of PtrCCR2, the only CCR member highly expressed in SDX, caused a reciprocal reduction in SDX protein CAD activities. The enzyme assays of mixed and coexpressed recombinant proteins supported physical interactions between PtrCAD1 and PtrCCR2. Biomolecular fluorescence complementation and pull-down/co-immunoprecipitation experiments supported a hypothesis of PtrCAD1/PtrCCR2 heterodimer formation. These results provide evidence for the formation of PtrCAD1/PtrCCR2 protein complexes in monolignol biosynthesis in planta.

Flux modeling for monolignol biosynthesis.

The pathway of monolignol biosynthesis involves many components interacting in a metabolic grid to regulate the supply and ratios of monolignols for lignification. The complexity of the pathway challenges any intuitive prediction of the output without mathematical modeling. Several models have been presented to quantify the metabolic flux for monolignol biosynthesis and the regulation of lignin content, composition, and structure in plant cell walls. Constraint-based models using data from transgenic plants were formulated to describe steady-state flux distribution in the pathway. Kinetic-based models using enzyme reaction and inhibition constants were developed to predict flux dynamics for monolignol biosynthesis in wood-forming cells. This review summarizes the recent progress in flux modeling and its application to lignin engineering for improved plant development and utilization.

Enzyme-Enzyme Interactions in Monolignol Biosynthesis.

The enzymes that comprise the monolignol biosynthetic pathway have been studied intensively for more than half a century. A major interest has been the role of pathway in the biosynthesis of lignin and the role of lignin in the formation of wood. The pathway has been typically conceived as linear steps that convert phenylalanine into three major monolignols or as a network of enzymes in a metabolic grid. Potential interactions of enzymes have been investigated to test models of metabolic channeling or for higher order interactions. Evidence for enzymatic or physical interactions has been fragmentary and limited to a few enzymes studied in different species. Only recently the entire pathway has been studied comprehensively in any single plant species. Support for interactions comes from new studies of enzyme activity, co-immunoprecipitation, chemical crosslinking, bimolecular fluorescence complementation, yeast 2-hybrid functional screening, and cell type-specific gene expression based on light amplification by stimulated emission of radiation capture microdissection. The most extensive experiments have been done on differentiating xylem of Populus trichocarpa, where genomic, biochemical, chemical, and cellular experiments have been carried out. Interactions affect the rate, direction, and specificity of both 3 and 4-hydroxylation in the monolignol biosynthetic pathway. Three monolignol P450 mono-oxygenases form heterodimeric and heterotetrameric protein complexes that activate specific hydroxylation of cinnamic acid derivatives. Other interactions include regulatory kinetic control of 4-coumarate CoA ligases through subunit specificity and interactions between a cinnamyl alcohol dehydrogenase and a cinnamoyl-CoA reductase. Monolignol enzyme interactions with other pathway proteins have been associated with biotic and abiotic stress response. Evidence challenging or supporting metabolic channeling in this pathway will be discussed.

Reciprocal cross-regulation of VND and SND multigene TF families for wood formation in Populus trichocarpa.

Secondary cell wall (SCW) biosynthesis is the biological process that generates wood, an important renewable feedstock for materials and energy. NAC domain transcription factors, particularly Vascular-Related NAC-Domain (VND) and Secondary Wall-Associated NAC Domain (SND) proteins, are known to regulate SCW differentiation. The regulation of VND and SND is important to maintain homeostasis for plants to avoid abnormal growth and development. We previously identified a splice variant, PtrSND1-A2IR , derived from PtrSND1-A2 as a dominant-negative regulator, which suppresses the transactivation of all PtrSND1 family members. PtrSND1-A2IR also suppresses the self-activation of the PtrSND1 family members except for its cognate transcription factor, PtrSND1-A2, suggesting the existence of an unknown factor needed to regulate PtrSND1-A2 Here, a splice variant, PtrVND6-C1IR , derived from PtrVND6-C1 was discovered that suppresses the protein functions of all PtrVND6 family members. PtrVND6-C1IR also suppresses the expression of all PtrSND1 members, including PtrSND1-A2, demonstrating that PtrVND6-C1IR is the previously unidentified regulator of PtrSND1-A2 We also found that PtrVND6-C1IR cannot suppress the expression of its cognate transcription factor, PtrVND6-C1PtrVND6-C1 is suppressed by PtrSND1-A2IR Both PtrVND6-C1IR and PtrSND1-A2IR cannot suppress their cognate transcription factors but can suppress all members of the other family. The results indicate that the splice variants from the PtrVND6 and PtrSND1 family may exert reciprocal cross-regulation for complete transcriptional regulation of these two families in wood formation. This reciprocal cross-regulation between families suggests a general mechanism among NAC domain proteins and likely other transcription factors, where intron-retained splice variants provide an additional level of regulation.

Tissue and cell-type co-expression networks of transcription factors and wood component genes in Populus trichocarpa.

MAIN CONCLUSION: Co-expression networks based on transcriptomes of Populus trichocarpa major tissues and specific cell types suggest redundant control of cell wall component biosynthetic genes by transcription factors in wood formation. We analyzed the transcriptomes of five tissues (xylem, phloem, shoot, leaf, and root) and two wood forming cell types (fiber and vessel) of Populus trichocarpa to assemble gene co-expression subnetworks associated with wood formation. We identified 165 transcription factors (TFs) that showed xylem-, fiber-, and vessel-specific expression. Of these 165 TFs, 101 co-expressed (correlation coefficient, r > 0.7) with the 45 secondary cell wall cellulose, hemicellulose, and lignin biosynthetic genes. Each cell wall component gene co-expressed on average with 34 TFs, suggesting redundant control of the cell wall component gene expression. Co-expression analysis showed that the 101 TFs and the 45 cell wall component genes each has two distinct groups (groups 1 and 2), based on their co-expression patterns. The group 1 TFs (44 members) are predominantly xylem and fiber specific, and are all highly positively co-expressed with the group 1 cell wall component genes (30 members), suggesting their roles as major wood formation regulators. Group 1 TFs include a lateral organ boundary domain gene (LBD) that has the highest number of positively correlated cell wall component genes (36) and TFs (47). The group 2 TFs have 57 members, including 14 vessel-specific TFs, and are generally less correlated with the cell wall component genes. An exception is a vessel-specific basic helix-loop-helix (bHLH) gene that negatively correlates with 20 cell wall component genes, and may function as a key transcriptional suppressor. The co-expression networks revealed here suggest a well-structured transcriptional homeostasis for cell wall component biosynthesis during wood formation.