Engineering custom morpho- and chemotypes of Populus for sustainable production of biofuels, bioproducts, and biomaterials.

Humans have been modifying plant traits for thousands of years, first through selection (i.e., domestication) then modern breeding, and in the last 30 years, through biotechnology. These modifications have resulted in increased yield, more efficient agronomic practices, and enhanced quality traits. Precision knowledge of gene regulation and function through high-resolution single-cell omics technologies, coupled with the ability to engineer plant genomes at the DNA sequence, chromatin accessibility, and gene expression levels, can enable engineering of complex and complementary traits at the biosystem level. Populus spp., the primary genetic model system for woody perennials, are among the fastest growing trees in temperate zones and are important for both carbon sequestration and global carbon cycling. Ample genomic and transcriptomic resources for poplar are available including emerging single-cell omics datasets. To expand use of poplar outside of valorization of woody biomass, chassis with novel morphotypes in which stem branching and tree height are modified can be fabricated thereby leading to trees with altered leaf to wood ratios. These morphotypes can then be engineered into customized chemotypes that produce high value biofuels, bioproducts, and biomaterials not only in specific organs but also in a cell-type-specific manner. For example, the recent discovery of triterpene production in poplar leaf trichomes can be exploited using cell-type specific regulatory sequences to synthesize high value terpenes such as the jet fuel precursor bisabolene specifically in the trichomes. By spatially and temporally controlling expression, not only can pools of abundant precursors be exploited but engineered molecules can be sequestered in discrete cell structures in the leaf. The structural diversity of the hemicellulose xylan is a barrier to fully utilizing lignocellulose in biomaterial production and by leveraging cell-type-specific omics data, cell wall composition can be modified in a tailored and targeted specific manner to generate poplar wood with novel chemical features that are amenable for processing or advanced manufacturing. Precision engineering poplar as a multi-purpose sustainable feedstock highlights how genome engineering can be used to re-imagine a crop species.

Plant Promoters and Terminators for High-Precision Bioengineering.

High-precision bioengineering and synthetic biology require fine-tuning gene expression at both transcriptional and posttranscriptional levels. Gene transcription is tightly regulated by promoters and terminators. Promoters determine the timing, tissues and cells, and levels of the expression of genes. Terminators mediate transcription termination of genes and affect mRNA levels posttranscriptionally, e.g., the 3'-end processing, stability, translation efficiency, and nuclear to cytoplasmic export of mRNAs. The promoter and terminator combination affects gene expression. In the present article, we review the function and features of plant core promoters, proximal and distal promoters, and terminators, and their effects on and benchmarking strategies for regulating gene expression.

Haplotype-resolved genome assembly of Populus tremula × P. alba reveals aspen-specific megabase satellite DNA.

Populus species play a foundational role in diverse ecosystems and are important renewable feedstocks for bioenergy and bioproducts. Hybrid aspen Populus tremula × P. alba INRA 717-1B4 is a widely used transformation model in tree functional genomics and biotechnology research. As an outcrossing interspecific hybrid, its genome is riddled with sequence polymorphisms which present a challenge for sequence-sensitive analyses. Here we report a telomere-to-telomere genome for this hybrid aspen with two chromosome-scale, haplotype-resolved assemblies. We performed a comprehensive analysis of the repetitive landscape and identified both tandem repeat array-based and array-less centromeres. Unexpectedly, the most abundant satellite repeats in both haplotypes lie outside of the centromeres, consist of a 147 bp monomer PtaM147, frequently span >1 megabases, and form heterochromatic knobs. PtaM147 repeats are detected exclusively in aspens (section Populus) but PtaM147-like sequences occur in LTR-retrotransposons of closely related species, suggesting their origin from the retrotransposons. The genomic resource generated for this transformation model genotype has greatly improved the design and analysis of genome editing experiments that are highly sensitive to sequence polymorphisms. The work should motivate future hypothesis-driven research to probe into the function of the abundant and aspen-specific PtaM147 satellite DNA.

Expression quantitative trait loci mapping identified PtrXB38 as a key hub gene in adventitious root development in Populus.

Plant establishment requires the formation and development of an extensive root system with architecture modulated by complex genetic networks. Here, we report the identification of the PtrXB38 gene as an expression quantitative trait loci (eQTL) hotspot, mapped using 390 leaf and 444 xylem Populus trichocarpa transcriptomes. Among predicted targets of this trans-eQTL were genes involved in plant hormone responses and root development. Overexpression of PtrXB38 in Populus led to significant increases in callusing and formation of both stem-born roots and base-born adventitious roots. Omics studies revealed that genes and proteins controlling auxin transport and signaling were involved in PtrXB38-mediated adventitious root formation. Protein-protein interaction assays indicated that PtrXB38 interacts with components of endosomal sorting complexes required for transport machinery, implying that PtrXB38-regulated root development may be mediated by regulating endocytosis pathway. Taken together, this work identified a crucial root development regulator and sheds light on the discovery of other plant developmental regulators through combining eQTL mapping and omics approaches.

Multiplex Editing of the Nucleoredoxin1 Tandem Array in Poplar: From Small Indels to Translocations and Complex Inversions.

The CRISPR-Cas9 system has been deployed for precision mutagenesis in an ever-growing number of species, including agricultural crops and forest trees. Its application to closely linked genes with extremely high sequence similarities has been less explored. In this study, we used CRISPR-Cas9 to mutagenize a tandem array of seven Nucleoredoxin1 (NRX1) genes spanning ∼100 kb in Populus tremula × Populus alba. We demonstrated efficient multiplex editing with one single guide RNA in 42 transgenic lines. The mutation profiles ranged from small insertions and deletions and local deletions in individual genes to large genomic dropouts and rearrangements spanning tandem genes. We also detected complex rearrangements including translocations and inversions resulting from multiple cleavage and repair events. Target capture sequencing was instrumental for unbiased assessments of repair outcomes to reconstruct unusual mutant alleles. The work highlights the power of CRISPR-Cas9 for multiplex editing of tandemly duplicated genes to generate diverse mutants with structural and copy number variations to aid future functional characterization.

Endogenous salicylic acid suppresses de novo root regeneration from leaf explants.

Plants can regenerate new organs from damaged or detached tissues. In the process of de novo root regeneration (DNRR), adventitious roots are frequently formed from the wound site on a detached leaf. Salicylic acid (SA) is a key phytohormone regulating plant defenses and stress responses. The role of SA and its acting mechanisms during de novo organogenesis is still unclear. Here, we found that endogenous SA inhibited the adventitious root formation after cutting. Free SA rapidly accumulated at the wound site, which was accompanied by an activation of SA response. SA receptors NPR3 and NPR4, but not NPR1, were required for DNRR. Wounding-elevated SA compromised the expression of AUX1, and subsequent transport of auxin to the wound site. A mutation in AUX1 abolished the enhanced DNRR in low SA mutants. Our work elucidates a role of SA in regulating DNRR and suggests a potential link between biotic stress and tissue regeneration.

The diversity of salicylic acid biosynthesis and defense signaling in plants: Knowledge gaps and future opportunities.

The phytohormone salicylic acid (SA) is known to regulate plant immunity against pathogens. Plants synthesize SA via the isochorismate synthase (ICS) pathway or the phenylalanine ammonia-lyase (PAL) pathway. The ICS pathway has been fully characterized using Arabidopsis thaliana, a model plant that exhibits pathogen-inducible SA accumulation. Many species including Populus (poplar) depend instead on the partially understood PAL pathway for constitutive as well as pathogen-stimulated SA synthesis. Diversity of SA-mediated defense is also evident in SA accumulation, redox regulation, and interplay with other hormones like jasmonic acid. This review highlights the contrast between Arabidopsis and poplar, discusses potential drivers of SA diversity in plant defenses, and offers future research directions.

Tonoplast Sucrose Trafficking Modulates Starch Utilization and Water Deficit Behavior in Poplar Leaves.

Leaf osmotic adjustment by the active accrual of compatible organic solutes (e.g. sucrose) contributes to drought tolerance throughout the plant kingdom. In Populus tremula x alba, PtaSUT4 encodes a tonoplast sucrose-proton symporter, whose downregulation by chronic mild drought or transgenic manipulation is known to increase leaf sucrose and turgor. While this may constitute a single drought tolerance mechanism, we now report that other adjustments which can occur during a worsening water deficit are damped when PtaSUT4 is constitutively downregulated. Specifically, we report that starch use and leaf relative water content (RWC) dynamics were compromised when plants with constitutively downregulated PtaSUT4 were subjected to a water deficit. Leaf RWC decreased more in wild-type and vector control lines than in transgenic PtaSUT4-RNAi (RNA-interference) or CRISPR (clustered regularly interspersed short palindromic repeats) knockout (KO) lines. The control line RWC decrease was accompanied by increased PtaSUT4 transcript levels and a mobilization of sucrose from the mesophyll-enriched leaf lamina into the midvein. The findings suggest that changes in SUT4 expression can increase turgor or decrease RWC as different tolerance mechanisms to reduced water availability. Evidence is presented that PtaSUT4-mediated sucrose partitioning between the vacuole and the cytosol is important not only for overall sucrose abundance and turgor, but also for reactive oxygen species (ROS) and antioxidant dynamics. Interestingly, the reduced capacity for accelerated starch breakdown under worsening water-deficit conditions was correlated with reduced ROS in the RNAi and KO lines. A role for PtaSUT4 in the orchestration of ROS, antioxidant, starch utilization and RWC dynamics during water stress and its importance in trees especially, with their high hydraulic resistances, is considered.

Lack of antagonism between salicylic acid and jasmonate signalling pathways in poplar.

Salicylic acid (SA) and jasmonic acid (JA) often play distinct roles in plant defence against pathogens. Research from Arabidopsis thaliana has established that SA- and JA-mediated defences are more effective against biotrophs and necrotrophs, respectively. These two hormones often interact antagonistically in response to particular attackers, with the induction of one leading to suppression of the other. Here, we report a contrasting pattern in the woody perennial Populus: positive SA-JA interplay. Using genetically engineered high SA lines of black poplar and wild-type lines after exogenous hormone application, we quantified SA and JA metabolites, signalling gene transcripts, antifungal flavonoids and resistance to rust (Melampsora larici-populina). Salicylic acid and JA metabolites were induced concurrently upon rust infection in poplar genotypes with varying resistance levels. Analysis of SA-hyperaccumulating transgenic poplar lines showed increased jasmonate levels, elevated flavonoid content and enhanced rust resistance, but no discernible reduction in growth. Exogenous application of either SA or JA triggered the accumulation of the other hormone. Expression of pathogenesis-related (PR) genes, frequently used as markers for SA signalling, was not correlated with SA content, but rather activated in proportion to pathogen infection. We conclude that SA and JA pathways interact positively in poplar resulting in the accumulation of flavonoid phytoalexins.

Multiplex knockout of trichome-regulating MYB duplicates in hybrid poplar using a single gRNA.

As the focus for CRISPR/Cas-edited plants moves from proof-of-concept to real-world applications, precise gene manipulation will increasingly require concurrent multiplex editing for polygenic traits. A common approach for editing across multiple sites is to design one guide RNA (gRNA) per target; however, this complicates construct assembly and increases the possibility of off-target mutations. In this study, we utilized one gRNA to target MYB186, a known positive trichome regulator, as well as its paralogs MYB138 and MYB38 at a consensus site for mutagenesis in hybrid poplar (Populus tremula × P. alba INRA 717-1B4). Unexpected duplications of MYB186 and MYB138 resulted in eight alleles for the three targeted genes in the hybrid poplar. Deep sequencing and polymerase chain reaction analyses confirmed editing across all eight targets in nearly all of the resultant glabrous mutants, ranging from small indels to large genomic dropouts, with no off-target activity detected at four potential sites. This highlights the effectiveness of a single gRNA targeting conserved exonic regions for multiplex editing. Additionally, cuticular wax and whole-leaf analyses showed a complete absence of triterpenes in the trichomeless mutants, hinting at a previously undescribed role for the nonglandular trichomes of poplar.

How do holoparasitic plants exploit vitamin K1?

Phylloquinone (vitamin K1) is a thylakoid-embedded electron carrier essential for photosynthesis. Paradoxically, we found that phylloquinone biosynthesis is retained in the nonphotosynthetic holoparasite Phelipanche aegyptiaca (Egyptian broomrape). The phylloquinone pathway genes are preferentially expressed during development of the invasive organ, the haustorium, and exhibit strong coexpression with redox-active proteins known to be involved in parasitism. Unlike in photoautotrophic taxa, the late pathway genes of the holoparasite lack the chloroplast-targeting sequence and their proteins are targeted to the plasma membrane instead. Plasma membrane phylloquinone may enable Phelipanche to sense changes in the redox environment during host interactions. The N-truncated isoforms are conserved in several other Orobanchaceae root holoparasites, and interestingly, in a number of closely related photoautotrophic species as well. This suggests an ancient origin of distinct phylloquinone pathways predating the evolution of parasitic plants in the Orobanchaceae. These findings represent exciting opportunities to probe plasma membrane phylloquinone function and diversification in parasitic and nonparasitic plant responses to external redox chemistry in the rhizosphere.

Synergies and Entanglement in Secondary Cell Wall Development and Abiotic Stress Response in Trees.

A major challenge for sustainable food, fuel, and fiber production is simultaneous genetic improvement of yield, biomass quality, and resilience to episodic environmental stress and climate change. For Populus and other forest trees, quality traits involve alterations in the secondary cell wall (SCW) of wood for traditional uses, as well as for a growing diversity of biofuels and bioproducts. Alterations in wood properties that are desirable for specific end uses can have negative effects on growth and stress tolerance. Understanding of the diverse roles of SCW genes is necessary for the genetic improvement of fast-growing, short-rotation trees that face perennial challenges in their growth and development. Here, we review recent progress into the synergies and antagonisms of SCW development and abiotic stress responses, particularly, the roles of transcription factors, SCW biogenesis genes, and paralog evolution.

Plasma membrane phylloquinone biosynthesis in nonphotosynthetic parasitic plants.

Nonphotosynthetic holoparasites exploit flexible targeting of phylloquinone biosynthesis to facilitate plasma membrane redox signaling. Phylloquinone is a lipophilic naphthoquinone found predominantly in chloroplasts and best known for its function in photosystem I electron transport and disulfide bridge formation of photosystem II subunits. Phylloquinone has also been detected in plasma membrane (PM) preparations of heterotrophic tissues with potential transmembrane redox function, but the molecular basis for this noncanonical pathway is unknown. Here, we provide evidence of PM phylloquinone biosynthesis in a nonphotosynthetic holoparasite Phelipanche aegyptiaca. A nonphotosynthetic and nonplastidial role for phylloquinone is supported by transcription of phylloquinone biosynthetic genes during seed germination and haustorium development, by PM-localization of alternative terminal enzymes, and by detection of phylloquinone in germinated seeds. Comparative gene network analysis with photosynthetically competent parasites revealed a bias of P. aegyptiaca phylloquinone genes toward coexpression with oxidoreductases involved in PM electron transport. Genes encoding the PM phylloquinone pathway are also present in several photoautotrophic taxa of Asterids, suggesting an ancient origin of multifunctionality. Our findings suggest that nonphotosynthetic holoparasites exploit alternative targeting of phylloquinone for transmembrane redox signaling associated with parasitism.

Defoliation-induced compensatory transpiration is compromised in SUT4-RNAi Populus.

The tonoplast sucrose transporter PtaSUT4 is well expressed in leaves of Populus tremula × Populus alba (INRA 717-IB4), and its inhibition by RNA-interference (RNAi) alters leaf sucrose homeostasis. Whether sucrose partitioning between the vacuole and the cytosol is modulated by PtaSUT4 for specific physiological outcomes in Populus remains unexplored. In this study, partial defoliation was used to elicit compensatory increases in photosynthesis and transpiration by the remaining leaves in greenhouse-grown poplar. Water uptake, leaf gas exchange properties, growth and nonstructural carbohydrate abundance in source and sink organs were then compared between wild-type and SUT4-RNAi lines. Partial defoliation increased maximum photosynthesis rates similarly in all lines. There was no indication that source leaf sugar levels changed differently between wild-type and RNAi plants following partial defoliation. Sink levels of hexose (glucose and fructose) and starch decreased similarly in all lines. Interestingly, plant water uptake after partial defoliation was not as well sustained in RNAi as in wild-type plants. While the compensatory increase in photosynthesis was similar between genotypes, leaf transpiration increased less robustly in RNAi than wild-type plants. SUT4-RNAi and wild-type source leaves differed constitutively in their bulk modulus of elasticity, a measure of leaf turgor, and storage water capacitance. The data demonstrate that reduced sucrose partitioning due to PtaSUT4-RNAi altered turgor control and compensatory transpiration capacity more strikingly than photosynthesis and sugar export. The results are consistent with the interpretation that SUT4 may control vacuolar turgor independently of sink carbon provisioning.

Compensatory Guaiacyl Lignin Biosynthesis at the Expense of Syringyl Lignin in 4CL1-Knockout Poplar.

The lignin biosynthetic pathway is highly conserved in angiosperms, yet pathway manipulations give rise to a variety of taxon-specific outcomes. Knockout of lignin-associated 4-coumarate:CoA ligases (4CLs) in herbaceous species mainly reduces guaiacyl (G) lignin and enhances cell wall saccharification. Here we show that CRISPR-knockout of 4CL1 in poplar (Populus tremula × alba) preferentially reduced syringyl (S) lignin, with negligible effects on biomass recalcitrance. Concordant with reduced S-lignin was downregulation of ferulate 5-hydroxylases (F5Hs). Lignification was largely sustained by 4CL5, a low-affinity paralog of 4CL1 typically with only minor xylem expression or activity. Levels of caffeate, the preferred substrate of 4CL5, increased in line with significant upregulation of caffeoyl shikimate esterase1 Upregulation of caffeoyl-CoA O-methyltransferase1 and downregulation of F5Hs are consistent with preferential funneling of 4CL5 products toward G-lignin biosynthesis at the expense of S-lignin. Thus, transcriptional and metabolic adaptations to 4CL1-knockout appear to have enabled 4CL5 catalysis at a level sufficient to sustain lignification. Finally, genes involved in sulfur assimilation, the glutathione-ascorbate cycle, and various antioxidant systems were upregulated in the mutants, suggesting cascading responses to perturbed thioesterification in lignin biosynthesis.

Shared expression of crassulacean acid metabolism (CAM) genes pre-dates the origin of CAM in the genus Yucca.

Crassulacean acid metabolism (CAM) is a carbon-concentrating mechanism that has evolved numerous times across flowering plants and is thought to be an adaptation to water-limited environments. CAM has been investigated from physiological and biochemical perspectives, but little is known about how plants evolve from C3 to CAM at the genetic or metabolic level. Here we take a comparative approach in analyzing time-course data of C3, CAM, and C3+CAM intermediate Yucca (Asparagaceae) species. RNA samples were collected over a 24 h period from both well-watered and drought-stressed plants, and were clustered based on time-dependent expression patterns. Metabolomic data reveal differences in carbohydrate metabolism and antioxidant response between the CAM and C3 species, suggesting that changes to metabolic pathways are important for CAM evolution and function. However, all three species share expression profiles of canonical CAM pathway genes, regardless of photosynthetic pathway. Despite differences in transcript and metabolite profiles between the C3 and CAM species, shared time-structured expression of CAM genes in both CAM and C3Yucca species suggests that ancestral expression patterns required for CAM may have pre-dated its origin in Yucca.

Salicylic acid activates poplar defense against the biotrophic rust fungus Melampsora larici-populina via increased biosynthesis of catechin and proanthocyanidins.

Poplar trees synthesize flavan-3-ols (catechin and proanthocyanidins) as a defense against foliar rust fungi, but the regulation of this defense response is poorly understood. Here, we investigated the role of hormones in regulating flavan-3-ol accumulation in poplar during rust infection. We profiled levels of defense hormones, signaling genes, and flavan-3-ol metabolites in black poplar leaves at different stages of rust infection. Hormone levels were manipulated by external sprays, genetic engineering, and drought to reveal their role in rust fungal defenses. Levels of salicylic acid (SA), jasmonic acid, and abscisic acid increased in rust-infected leaves and activated downstream signaling, with SA levels correlating closely with those of flavan-3-ols. Pretreatment with the SA analog benzothiadiazole increased flavan-3-ol accumulation by activating the MYB-bHLH-WD40 complex and reduced rust proliferation. Furthermore, transgenic poplar lines overproducing SA exhibited higher amounts of flavan-3-ols constitutively via the same transcriptional activation mechanism. These findings suggest a strong association among SA, flavan-3-ol biosynthesis, and rust resistance in poplars. Abscisic acid also promoted poplar defense against rust infection, but likely through stomatal immunity independent of flavan-3-ols. Jasmonic acid did not confer any apparent defense responses to the fungal pathogen. We conclude that SA activates flavan-3-ol biosynthesis in poplar against rust infection.

Genome Editing in Trees: From Multiple Repair Pathways to Long-Term Stability.

The CRISPR technology continues to diversify with a broadening array of applications that touch all kingdoms of life. The simplicity, versatility and species-independent nature of the CRISPR system offers researchers a previously unattainable level of precision and control over genomic modifications. Successful applications in forest, fruit and nut trees have demonstrated the efficacy of CRISPR technology at generating null mutations in the first generation. This eliminates the lengthy process of multigenerational crosses to obtain homozygous knockouts (KO). The high degree of genome heterozygosity in outcrossing trees is both a challenge and an opportunity for genome editing: a challenge because sequence polymorphisms at the target site can render CRISPR editing ineffective; yet an opportunity because the power and specificity of CRISPR can be harnessed for allele-specific editing. Examination of CRISPR/Cas9-induced mutational profiles from published tree studies reveals the potential involvement of multiple DNA repair pathways, suggesting that the influence of sequence context at or near the target sites can define mutagenesis outcomes. For commercial production of elite trees that rely on vegetative propagation, available data suggest an excellent outlook for stable CRISPR-induced mutations and associated phenotypes over multiple clonal generations.

Host-plant induced changes in microbial community structure and midgut gene expression in an invasive polyphage (Anoplophora glabripennis).

Polyphagous insect herbivores possess diverse mechanisms to overcome challenges of feeding in multiple plant species including, but not limited to, transcriptional plasticity and associations with obligate or facultative symbionts. The Asian longhorned beetle (Anoplophora glabripennis) is a polyphagous wood-feeder capable of developing on over 100 tree species and, like other polyphages, its genome contains amplifications of digestive and detoxification genes. This insect also possesses a diverse gut microbial community, which has the metabolic potential to augment digestive physiology. While the genomic repertoires of A. glabripennis and its microbial community have been studied previously, comparatively less is known about how the gut transcriptome and community change in response to feeding in different hosts. In this study, we show that feeding in two suitable hosts (Acer spp. and Populus nigra) altered the expression levels of multicopy genes linked to digestion and detoxification. However, feeding in a host with documented resistance (Populus tomentosa) induced changes in the transcriptome and community beyond what was observed in insects reared in P. nigra, including the downregulation of numerous ß-glucosidases, odorant binding proteins, and juvenile hormone binding proteins, the upregulation of several cuticular genes, and the loss of one major bacterial family from the gut community.