The transcriptional regulation of a putative hemicellulose gene, PtrPARVUS2 in poplar.

The plant cell wall serves as a critical interface between the plant and its environment, offering protection against various stresses and contributing to biomass production. Hemicellulose is one of the major components of the cell wall, and understanding the transcriptional regulation of its production is essential to fully understanding cell wall formation. This study explores the regulatory mechanisms underlying one of the genes involved in hemicellulose biosynthesis, PtrPARVUS2. Six transcription factors (TFs) were identified from a xylem-biased library to negatively regulate PtrPARVUS2 expression. These TFs, belonging to diverse TF families, were confirmed to bind to specific cis-elements in the PtrPARVUS2 promoter region, as validated by Yeast One-Hybrid (Y1H) assays, transient expression analysis, and Chromatin Immunoprecipitation sequencing (ChIP-seq) assays. Furthermore, motif analysis identified putative cis-regulatory elements bound by these TFs, shedding light on the transcriptional regulation of SCW biosynthesis genes. Notably, several TFs targeted genes encoding uridine diphosphate glycosyltransferases (UGTs), crucial enzymes involved in hemicellulose glycosylation. Phylogenetic analysis of UGTs regulated by these TFs highlighted their diverse roles in modulating hemicellulose synthesis. Overall, this study identifies a set of TFs that regulate PARVUS2 in poplar, providing insights into the intricate coordination of TFs and PtrPARVUS2 in SCW formation. Understanding these regulatory mechanisms enhances our ability to engineer plant biomass for tailored applications, including biofuel production and bioproduct development.

Functional Diversification and the Plant Secondary Cell Wall.

Much evidence exists suggesting the presence of genetic functional diversification in plants, though literature associated with the role of functional diversification in the evolution of the plant secondary cell wall (SCW) has sparsely been compiled and reviewed in a recent context. This review aims to elucidate, through the examination of gene phylogenies associated with its biosynthesis and maintenance, the role of functional diversification in shaping the critical, dynamic, and characteristic organelle, the secondary cell wall. It will be asserted that gene families resulting from gene duplication and subsequent functional divergence are present and are heavily involved in SCW biosynthesis and maintenance. Furthermore, diversification will be presented as a significant driver behind the evolution of the many functional characteristics of the SCW. The structure and function of the plant cell wall and its constituents will first be explored, followed by a discussion on the phenomenon of gene duplication and the resulting genetic functional divergence that can emerge. Finally, the major constituents of the SCW and their individual relationships with duplication and divergence will be reviewed to the extent of current knowledge on the subject.

Nutrient conditions mediate mycorrhizal effects on biomass production and cell wall chemistry in poplar.

Large-scale biofuel production from lignocellulosic feedstock is limited by the financial and environmental costs associated with growing and processing lignocellulosic material and the resilience of these plants to environmental stress. Symbiotic associations with arbuscular (AM) and ectomycorrhizal (EM) fungi represent a potential strategy for expanding feedstock production while reducing nutrient inputs. Comparing AM and EM effects on wood production and chemical composition is a necessary step in developing biofuel feedstocks. Here, we assessed the productivity, biomass allocation and secondary cell wall (SCW) composition of greenhouse-grown Populus tremuloidesMichx. inoculated with either AM or EM fungi. Given the long-term goal of reducing nutrient inputs for biofuel production, we further tested the effects of nutrient availability and nitrogen:phosphorus stoichiometry on mycorrhizal responses. Associations with both AM and EM fungi increased plant biomass by 14-74% depending on the nutrient conditions but had minimal effects on SCW composition. Mycorrhizal plants, especially those inoculated with EM fungi, also allocated a greater portion of their biomass to roots, which could be beneficial in the field where plants are likely to experience both water and nutrient stress. Leaf nutrient content was weakly but positively correlated with wood production in mycorrhizal plants. Surprisingly, phosphorus played a larger role in EM plants compared with AM plants. Relative nitrogen and phosphorus availability were correlated with shifts in SCW composition. For AM associations, the benefit of increased wood biomass may be partially offset by increased lignin content, a trait that affects downstream processing of lignocellulosic tissue for biofuels. By comparing AM and EM effects on the productivity and chemical composition of lignocellulosic tissue, this work links broad functional diversity in mycorrhizal associations to key biofuel traits and highlights the importance of considering both biotic and abiotic factors when developing strategies for sustainable biofuel production.

Pectin and Xylan Biosynthesis in Poplar: Implications and Opportunities for Biofuels Production.

A potential method by which society's reliance on fossil fuels can be lessened is via the large-scale utilization of biofuels derived from the secondary cell walls of woody plants; however, there remain a number of technical challenges to the large-scale production of biofuels. Many of these challenges emerge from the underlying complexity of the secondary cell wall. The challenges associated with lignin have been well explored elsewhere, but the dicot cell wall components of hemicellulose and pectin also present a number of difficulties. Here, we provide an overview of the research wherein pectin and xylan biosynthesis has been altered, along with investigations on the function of irregular xylem 8 (IRX8) and glycosyltransferase 8D (GT8D), genes putatively involved in xylan and pectin synthesis. Additionally, we provide an analysis of the evidence in support of two hypotheses regarding GT8D and conclude that while there is evidence to lend credence to these hypotheses, there are still questions that require further research and examination.

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.

Expression of a hyperthermophilic endoglucanase in hybrid poplar modifies the plant cell wall and enhances digestibility.

BACKGROUND: Expression of glycosyl hydrolases in lignocellulosic biomass has been proposed as an alternative to improve efficiency of cellulosic ethanol production. In planta production of hyperthermophilic hydrolytic enzymes could prevent the detrimental effects often seen resulting from the expression of recombinant mesophilic enzymes to plant hosts. Utilizing lignocellulosic feedstocks to produce hyperthermophilic hydrolases provides additional benefits for ethanol production in the way of transgenic feedstocks serving as both enzyme providers and cellulosic substrates. RESULTS: In this study, transgenic hybrid poplar (Populus alba × grandidentata) was generated to express a hyperthermophilic endoglucanase from Thermotoga neapolitana with an optimal temperature over 100 °C. Functional hyperthermoactive endoglucanase was successfully produced in the transgenic events, and altered phenotypic growth was observed in transgenic lines. Moreover, the line with the highest TnCelB expression in both leaf and developing xylem had reduced lignin content and cellulose crystallinity, resulting in a more digestible cell wall. The activation of TnCelB by a post-harvest heat treatment resulted in enhanced saccharification efficiencies of transgenic poplar lines with moderate TnCelB expression and without alteration of cellulose and lignin when not heat-treated. In planta high-level overexpression of a hyperthermophilic endoglucanase paired with heat treatment following harvest, resulted in biomass that was comparable with wild-type lines that underwent a traditional pretreatment for saccharification. CONCLUSIONS: Overexpression of hyperthermophilic endoglucanase in feedstock had impacts on plant growth and cell wall composition, especially when the enzyme was highly expressed. Improved glucan saccharification efficiencies from transgenic lines before and after heat treatment could reduce both the economic and environmental costs associated with ethanol production from lignocellulosic biomass.

Cell wall composition and lignin biosynthetic gene expression along a developmental gradient in an Australian sugarcane cultivar.

Sugarcane bagasse is an abundant source of lignocellulosic material for bioethanol production. Utilisation of bagasse for biofuel production would be environmentally and economically beneficial, but the recalcitrance of lignin continues to provide a challenge. Further understanding of lignin production in specific cultivars will provide a basis for modification of genomes for the production of phenotypes with improved processing characteristics. Here we evaluated the expression profile of lignin biosynthetic genes and the cell wall composition along a developmental gradient in KQ228 sugarcane. The expression levels of nine lignin biosynthesis genes were quantified in five stem sections of increasing maturity and in root tissue. Two distinct expression patterns were seen. The first saw highest gene expression in the youngest tissue, with expression decreasing as tissue matured. The second pattern saw little to no change in transcription levels across the developmental gradient. Cell wall compositional analysis of the stem sections showed total lignin content to be significantly higher in more mature tissue than in the youngest section assessed. There were no changes in structural carbohydrates across developmental sections. These gene expression and cell wall compositional patterns can be used, along with other work in grasses, to inform biotechnological approaches to crop improvement for lignocellulosic biofuel production.

RNAi downregulation of three key lignin genes in sugarcane improves glucose release without reduction in sugar production.

BACKGROUND: Sugarcane is a subtropical crop that produces large amounts of biomass annually. It is a key agricultural crop in many countries for the production of sugar and other products. Residual bagasse following sucrose extraction is currently underutilized and it has potential as a carbohydrate source for the production of biofuels. As with all lignocellulosic crops, lignin acts as a barrier to accessing the polysaccharides, and as such, is the focus of transgenic efforts. In this study, we used RNAi to individually reduce the expression of three key genes in the lignin biosynthetic pathway in sugarcane. These genes, caffeoyl-CoA O-methyltransferase (CCoAOMT), ferulate 5-hydroxylase (F5H) and caffeic acid O-methyltransferase (COMT), impact lignin content and/or composition. RESULTS: For each RNAi construct, we selected three events for further analysis based on qRT-PCR results. For the CCoAOMT lines, there were no lines with a reduction in lignin content and only one line showed improved glucose release. For F5H, no lines had reduced lignin, but one line had a significant increase in glucose release. For COMT, one line had reduced lignin content, and this line and another released higher levels of glucose during enzymatic hydrolysis. Two of the lines with improved glucose release (F5H-2 and COMT-2) also had reduced S:G ratios. CONCLUSIONS: Along with improvements in bagasse quality for the production of lignocellulosic-based fuels, there was only one line with reduction in juice sucrose extraction, and three lines with significantly improved sucrose production, providing evidence that the alteration of sugarcane for improved lignocellulosic ethanol production can be achieved without negatively impacting sugar production and perhaps even enhancing it.

Sugarcane transgenics expressing MYB transcription factors show improved glucose release.

BACKGROUND: Sugarcane, a tropical C4 perennial crop, is capable of producing 30-100 tons or more of biomass per hectare annually. The lignocellulosic residue remaining after sugar extraction is currently underutilized and can provide a significant source of biomass for the production of second-generation bioethanol. RESULTS: MYB31 and MYB42 were cloned from maize and expressed in sugarcane with and without the UTR sequences. The cloned sequences were 98 and 99 % identical to the published nucleotide sequences. The inclusion of the UTR sequences did not affect any of the parameters tested. There was little difference in plant height and the number of internodes of the MYB-overexpressing sugarcane plants when compared with controls. MYB transgene expression determined by qPCR exhibited continued expression in young and maturing internodes. MYB31 downregulated more genes within the lignin biosynthetic pathway than MYB42. MYB31 and MYB42 expression resulted in decreased lignin content in some lines. All MYB42 plants further analyzed showed significant increases in glucose release by enzymatic hydrolysis in 72 h, whereas only two MYB31 plants released more glucose than control plants. This correlated directly with a significant decrease in acid-insoluble lignin. Soluble sucrose content of the MYB42 transgenic plants did not vary compared to control plants. CONCLUSIONS: This study demonstrates the use of MYB transcription factors to improve the production of bioethanol from sugarcane bagasse remaining after sugar extraction.

Improved molecular tools for sugar cane biotechnology.

Sugar cane is a major source of food and fuel worldwide. Biotechnology has the potential to improve economically-important traits in sugar cane as well as diversify sugar cane beyond traditional applications such as sucrose production. High levels of transgene expression are key to the success of improving crops through biotechnology. Here we describe new molecular tools that both expand and improve gene expression capabilities in sugar cane. We have identified promoters that can be used to drive high levels of gene expression in the leaf and stem of transgenic sugar cane. One of these promoters, derived from the Cestrum yellow leaf curling virus, drives levels of constitutive transgene expression that are significantly higher than those achieved by the historical benchmark maize polyubiquitin-1 (Zm-Ubi1) promoter. A second promoter, the maize phosphonenolpyruvate carboxylate promoter, was found to be a strong, leaf-preferred promoter that enables levels of expression comparable to Zm-Ubi1 in this organ. Transgene expression was increased approximately 50-fold by gene modification, which included optimising the codon usage of the coding sequence to better suit sugar cane. We also describe a novel dual transcriptional enhancer that increased gene expression from different promoters, boosting expression from Zm-Ubi1 over eightfold. These molecular tools will be extremely valuable for the improvement of sugar cane through biotechnology.

An improved chemically inducible gene switch that functions in the monocotyledonous plant sugar cane.

Chemically inducible gene switches can provide precise control over gene expression, enabling more specific analyses of gene function and expanding the plant biotechnology toolkit beyond traditional constitutive expression systems. The alc gene expression system is one of the most promising chemically inducible gene switches in plants because of its potential in both fundamental research and commercial biotechnology applications. However, there are no published reports demonstrating that this versatile gene switch is functional in transgenic monocotyledonous plants, which include some of the most important agricultural crops. We found that the original alc gene switch was ineffective in the monocotyledonous plant sugar cane, and describe a modified alc system that is functional in this globally significant crop. A promoter consisting of tandem copies of the ethanol receptor inverted repeat binding site, in combination with a minimal promoter sequence, was sufficient to give enhanced sensitivity and significantly higher levels of ethanol inducible gene expression. A longer CaMV 35S minimal promoter than was used in the original alc gene switch also substantially improved ethanol inducibility. Treating the roots with ethanol effectively induced the modified alc system in sugar cane leaves and stem, while an aerial spray was relatively ineffective. The extension of this chemically inducible gene expression system to sugar cane opens the door to new opportunities for basic research and crop biotechnology.

Enhanced expression of glutamine synthetase (GS1a) confers altered fibre and wood chemistry in field grown hybrid poplar (Populus tremula X alba) (717-1B4).

Hybrid poplar (Populus tremula X P. alba) genetically engineered to express the pine cytosolic glutamine synthetase gene (GS1a) has been previously shown to display desirable field performance characteristics, including enhancements in growth and nitrogen use efficiency. Analysis of wood samples from a 3-year-old field trial of three independently transformed GS1a transgenic hybrid poplar lines revealed that, when compared with wild-type controls, ectopic expression of GS1a resulted in alterations in wood properties and wood chemistry. Included were significant enhancements in wood fibre length, wood density, microfibre angle, per cent syringyl lignin and elevated concentrations of wood sugars, specifically glucose, galactose, mannose and xylose. Total extractive content and acid-insoluble lignin were significantly reduced in wood of GS1a transgenics when compared with wild-type trees. Together, these cell wall characteristics resulted in improved wood pulping attributes, including improved lignin solubilization with no concurrent decrease in yield. Trees with increased GS1a expression have improved characteristics for pulp and paper production and hold potential as a feedstock for biofuels production.

Effects on Lignin Structure of Coumarate 3-Hydroxylase Downregulation in Poplar.

The lignin structural ramifications of coumarate 3-hydroxylase (C3H) downregulation have not been addressed in hardwoods. Such information is required to accompany an assessment of the digestibility and bioenergy performance characteristics of poplar, in particular. Structurally rich 2D NMR methods were applied to the entire lignin fraction to delineate lignin p-hydroxyphenyl:guaiacyl:syringyl (H:G:S) levels and linkage distribution changes (and to compare with traditional degradative analyses). C3H downregulation reduced lignin levels by half and markedly increased the proportion of H units relative to the normally dominant G and S units. Relative stem H unit levels were up by ∼ 100-fold to ∼ 31 %, almost totally at the expense of G units; differences in the lignin interunit linkage distributions were more subtle. The H level in the most drastically C3H-downregulated transgenic poplar falls well beyond the H:G:S compositional bounds of normal angiosperms. The response observed here, in poplar, differs markedly from that reported for alfalfa where the S:G ratio remained almost constant even at substantial H levels, highlighting the often differing responses among plant species.

Accumulation of recombinant cellobiohydrolase and endoglucanase in the leaves of mature transgenic sugar cane.

A major strategic goal in making ethanol from lignocellulosic biomass a cost-competitive liquid transport fuel is to reduce the cost of production of cellulolytic enzymes that hydrolyse lignocellulosic substrates to fermentable sugars. Current production systems for these enzymes, namely microbes, are not economic. One way to substantially reduce production costs is to express cellulolytic enzymes in plants at levels that are high enough to hydrolyse lignocellulosic biomass. Sugar cane fibre (bagasse) is the most promising lignocellulosic feedstock for conversion to ethanol in the tropics and subtropics. Cellulolytic enzyme production in sugar cane will have a substantial impact on the economics of lignocellulosic ethanol production from bagasse. We therefore generated transgenic sugar cane accumulating three cellulolytic enzymes, fungal cellobiohydrolase I (CBH I), CBH II and bacterial endoglucanase (EG), in leaves using the maize PepC promoter as an alternative to maize Ubi1 for controlling transgene expression. Different subcellular targeting signals were shown to have a substantial impact on the accumulation of these enzymes; the CBHs and EG accumulated to higher levels when fused to a vacuolar-sorting determinant than to an endoplasmic reticulum-retention signal, while EG was produced in the largest amounts when fused to a chloroplast-targeting signal. These results are the first demonstration of the expression and accumulation of recombinant CBH I, CBH II and EG in sugar cane and represent a significant first step towards the optimization of cellulolytic enzyme expression in sugar cane for the economic production of lignocellulosic ethanol.

Altered sucrose metabolism impacts plant biomass production and flower development.

Nicotiana tabacum (tobacco) was transformed with three genes involved in sucrose metabolism, UDP-glucose pyrophosphorylase (UGPase, EC 2.7.7.9), sucrose synthase (SuSy, EC 2.4.1.13) and sucrose phosphate synthase (SPS, EC 2.4.1.14). Plants harbouring the single transgenes were subsequently crossed to produce double and triple transgenic lines, including: 2 x 35S::UGPase x SPS, 4CL::UGPase x SPS, 2 x 35S::SuSy x SPS, 4CL::SuSy x SPS, 2 x 35S::UGPase x SuSy x SPS, and 4CL::UGPase x SuSy x SPS. The ultimate aim of the study was to examine whether it is possible to alter cellulose production through the manipulation of sucrose metabolism genes. While altering sucrose metabolism using UGPase, SuSy and SPS does not have an end effect on cellulose production, their simultaneous overexpression resulted in enhanced primary growth as seen in an increase in height growth, in some cases over 50%. Furthermore, the pyramiding strategy of simultaneously altering the expression of multiple genes in combination resulted in increased time to reproductive bud formation as well as altered flower morphology and foliar stipule formation in 4CL lines. Upregulation of these sucrose metabolism genes appears to directly impact primary growth and therefore biomass production in tobacco.

Sucrose synthase affects carbon partitioning to increase cellulose production and altered cell wall ultrastructure.

Overexpression of the Gossypium hirsutum sucrose synthase (SuSy) gene under the control of 2 promoters was examined in hybrid poplar (Populus alba x grandidentata). Analysis of RNA transcript abundance, enzyme activity, cell wall composition, and soluble carbohydrates revealed significant changes in the transgenic lines. All lines showed significantly increased SuSy enzyme activity in developing xylem. This activity manifested in altered secondary cell wall cellulose content per dry weight in all lines, with increases of 2% to 6% over control levels, without influencing plant growth. The elevated concentration of cellulose was associated with an increase in cell wall crystallinity but did not alter secondary wall microfibril angle. This finding suggests that the observed increase in crystallinity is a function of altered carbon partitioning to cellulose biosynthesis rather than the result of tension wood formation. Furthermore, the augmented deposition of cellulose in the transgenic lines resulted in thicker xylem secondary cell wall and consequently improved wood density. These findings clearly implicate SuSy as a key regulator of sink strength in poplar trees and demonstrate the tight association of SuSy with cellulose synthesis and secondary wall formation.

Perturbed lignification impacts tree growth in hybrid poplar--a function of sink strength, vascular integrity, and photosynthetic assimilation.

The effects of reductions in cell wall lignin content, manifested by RNA interference suppression of coumaroyl 3'-hydroxylase, on plant growth, water transport, gas exchange, and photosynthesis were evaluated in hybrid poplar trees (Populus alba x grandidentata). The growth characteristics of the reduced lignin trees were significantly impaired, resulting in smaller stems and reduced root biomass when compared to wild-type trees, as well as altered leaf morphology and architecture. The severe inhibition of cell wall lignification produced trees with a collapsed xylem phenotype, resulting in compromised vascular integrity, and displayed reduced hydraulic conductivity and a greater susceptibility to wall failure and cavitation. In the reduced lignin trees, photosynthetic carbon assimilation and stomatal conductance were also greatly reduced, however, shoot xylem pressure potential and carbon isotope discrimination were higher and water-use efficiency was lower, inconsistent with water stress. Reductions in assimilation rate could not be ascribed to increased stomatal limitation. Starch and soluble sugars analysis of leaves revealed that photosynthate was accumulating to high levels, suggesting that the trees with substantially reduced cell wall lignin were not carbon limited and that reductions in sink strength were, instead, limiting photosynthesis.

RNAi-mediated suppression of p-coumaroyl-CoA 3'-hydroxylase in hybrid poplar impacts lignin deposition and soluble secondary metabolism.

p-Coumaroyl-CoA 3'-hydroxylase (C3'H) is a cytochrome P450-dependent monooxygenase that catalyzes the 3'-hydroxylation of p-coumaroyl shikimate and p-coumaroyl quinate. We used RNA interference to generate transgenic hybrid poplar suppressed in C3'H expression and analyzed them with respect to transcript abundance, cell wall structure and chemical composition, and soluble metabolite levels. RT-PCR expression profiles confirmed the down-regulation of C3'H in a number of lines, which generally correlated very well with reduced total cell wall lignin content. The most strongly repressed line was chosen for further analysis and compared with the wild-type trees. In-depth characterization revealed that along with the significant decrease in total lignin content, a significant shift in lignin monomer composition was observed, favoring the generation of p-hydroxyphenyl units at the expense of guaiacyl units while the proportion of syringyl moieties remained constant. Suppression of C3'H also resulted in the accumulation of substantial pools of 1-O-p-coumaroyl-beta-d-glucoside and other phenylpropanoid glycosides, and p-coumaroyl shikimate, providing further insight into the role of C3'H in the lignin biosynthetic pathway. The data presented indicate that when down-regulated, C3'H becomes a rate-limiting step in lignin biosynthesis and further support the involvement of hydroxycinnamic acid shikimate esters in the lignin biosynthetic pathway.

Over-expression of UDP-glucose pyrophosphorylase in hybrid poplar affects carbon allocation.

The effects of the over-expression of the Acetobacter xylinum UDP-glucose pyrophosphorylase (UGPase) under the control of the tandem repeat Cauliflower Mosaic Virus promoter (2x35S) on plant metabolism and growth were investigated in hybrid poplar (Populus albaxgrandidentata). Transcript levels, enzyme activity, growth parameters, leaf morphology, structural and soluble carbohydrates, and soluble metabolite levels were quantified in both transgenic and wild-type trees. Transgenic 2x35S::UGPase poplar showed impaired growth rates, displaying reduced height growth and stem diameter. Morphologically, 2x35S::UGPase trees had elongated axial shoots, and leaves that were substantially smaller in size when compared with wild-type trees at equivalent developmental stages. Biochemical analysis revealed significant increases in soluble sugar, starch, and cellulose contents, and concurrent decreases in lignin content. Lignin monomer composition was altered in favour of syringyl moieties. Detailed soluble metabolite analysis revealed that 2x35S::UGPase trees had as much as a 270-fold increase in the salicylic acid 2-O-beta-D-glucoside (SAG), a compound typically associated with the stress response. These data suggest that while it is possible to alter the allocation of carbon in favour of cellulose biosynthesis, whole plant changes result in unexpected decreases in growth and an increase in defence metabolites.