Heterologous expression of Arabidopsis laccase2, laccase4 and peroxidase52 driven under developing xylem specific promoter DX15 improves saccharification in populus.

BACKGROUND: Secondary cell wall holds considerable potential as it has gained immense momentum to replace the lignocellulosic feedstock into fuels. Lignin one of the components of secondary cell wall tightly holds the polysaccharides thereby enhancing the recalcitrance and complexity in the biomass. Laccases (LAC) and peroxidases (PRX) are the major phenyl-oxidases playing key functions during the polymerization of monolignols into lignin. Yet, the functions of laccase and peroxidases gene families remained largely unknown. Hence, the objective of this conducted study is to understand the role of specific LAC and PRX in Populus wood formation and to further investigate how the altered Lac and Prx expression affects biomass recalcitrance and plant growth. This study of heterologous expression of Arabidopsis Lac and Prx genes was conducted in poplar to avoid any otherwise occurring co-suppression mechanism during the homologous overexpression of highly expressed native genes. In the pursuit of optimizing lignocellulosic biomass for biofuel production, the present study focuses on harnessing the enzymatic potential of Arabidopsis thaliana Laccase2, Laccase4, and Peroxidase52 through heterologous expression. RESULTS: We overexpressed selected Arabidopsis laccase2 (AtLac2), laccase4 (AtLac4), and peroxidase52 (AtPrx52) genes, based on their high transcript expression respective to the differentiating xylem tissues in the stem, in hybrid poplar (cv. 717) expressed under the developing xylem tissue-specific promoter, DX15 characterized the transgenic populus for the investigation of growth phenotypes and recalcitrance efficiency. Bioinformatics analyses conducted on AtLac2 and AtLac4 and AtPrx52, revealed the evolutionary relationship between the laccase gene and peroxidase gene homologs, respectively. Transgenic poplar plant lines overexpressing the AtLac2 gene (AtLac2-OE) showed an increase in plant height without a change in biomass yield as compared to the controls; whereas, AtLac4-OE and AtPrx52-OE transgenic lines did not show any such observable growth phenotypes compared to their respective controls. The changes in the levels of lignin content and S/G ratios in the transgenic poplar resulted in a significant increase in the saccharification efficiency as compared to the control plants. CONCLUSIONS: Overall, saccharification efficiency was increased by 35-50%, 21-42%, and 8-39% in AtLac2-OE, AtLac4-OE, and AtPrx52-OE transgenic poplar lines, respectively, as compared to their controls. Moreover, the bioengineered plants maintained normal growth and development, underscoring the feasibility of this approach for biomass improvement without compromising overall plant fitness. This study also sheds light on the potential of exploiting regulatory elements of DX15 to drive targeted expression of lignin-modifying enzymes, thereby providing a promising avenue for tailoring biomass for improved biofuel production. These findings contribute to the growing body of knowledge in synthetic biology and plant biotechnology, offering a sustainable solution to address the challenges associated with lignocellulosic biomass recalcitrance.

Understanding the Modus Operandi of Class II KNOX Transcription Factors in Secondary Cell Wall Biosynthesis.

Lignocellulosic biomass from the secondary cell walls of plants has a veritable potential to provide some of the most appropriate raw materials for producing second-generation biofuels. Therefore, we must first understand how plants synthesize these complex secondary cell walls that consist of cellulose, hemicellulose, and lignin in order to deconstruct them later on into simple sugars to produce bioethanol via fermentation. Knotted-like homeobox (KNOX) genes encode homeodomain-containing transcription factors (TFs) that modulate various important developmental processes in plants. While Class I KNOX TF genes are mainly expressed in the shoot apical meristems of both monocot and eudicot plants and are involved in meristem maintenance and/or formation, Class II KNOXTF genes exhibit diverse expression patterns and their precise functions have mostly remained unknown, until recently. The expression patterns of Class II KNOX TF genes in Arabidopsis, namely KNAT3, KNAT4, KNAT5, and KNAT7, suggest that TFs encoded by at least some of these genes, such as KNAT7 and KNAT3, may play a significant role in secondary cell wall formation. Specifically, the expression of the KNAT7 gene is regulated by upstream TFs, such as SND1 and MYB46, while KNAT7 interacts with other cell wall proteins, such as KNAT3, MYB75, OFPs, and BLHs, to regulate secondary cell wall formation. Moreover, KNAT7 directly regulates the expression of some xylan synthesis genes. In this review, we summarize the current mechanistic understanding of the roles of Class II KNOX TFs in secondary cell wall formation. Recent success with the genetic manipulation of Class II KNOX TFs suggests that this may be one of the biotechnological strategies to improve plant feedstocks for bioethanol production.

Genetic Modification of KNAT7 Transcription Factor Expression Enhances Saccharification and Reduces Recalcitrance of Woody Biomass in Poplars.

The precise role of KNAT7 transcription factors (TFs) in regulating secondary cell wall (SCW) biosynthesis in poplars has remained unknown, while our understanding of KNAT7 functions in other plants is continuously evolving. To study the impact of genetic modifications of homologous and heterologous KNAT7 gene expression on SCW formation in transgenic poplars, we prepared poplar KNAT7 (PtKNAT7) overexpression (PtKNAT7-OE) and antisense suppression (PtKNAT7-AS) vector constructs for the generation of transgenic poplar lines via Agrobacterium-mediated transformation. Since the overexpression of homologous genes can sometimes result in co-suppression, we also overexpressed Arabidopsis KNAT7 (AtKNAT7-OE) in transgenic poplars. In all these constructs, the expression of KNAT7 transgenes was driven by developing xylem (DX)-specific promoter, DX15. Compared to wild-type (WT) controls, many SCW biosynthesis genes downstream of KNAT7 were highly expressed in poplar PtKNAT7-OE and AtKNAT7-OE lines. Yet, no significant increase in lignin content of woody biomass of these transgenic lines was observed. PtKNAT7-AS lines, however, showed reduced expression of many SCW biosynthesis genes downstream of KNAT7 accompanied by a reduction in lignin content of wood compared to WT controls. Syringyl to Guaiacyl lignin (S/G) ratios were significantly increased in all three KNAT7 knockdown and overexpression transgenic lines than WT controls. These transgenic lines were essentially indistinguishable from WT controls in terms of their growth phenotype. Saccharification efficiency of woody biomass was significantly increased in all transgenic lines than WT controls. Overall, our results demonstrated that developing xylem-specific alteration of KNAT7 expression affects the expression of SCW biosynthesis genes, impacting at least the lignification process and improving saccharification efficiency, hence providing one of the powerful tools for improving bioethanol production from woody biomass of bioenergy crops and trees.

Temporal transcriptome profiling of developing seeds reveals a concerted gene regulation in relation to oil accumulation in Pongamia (Millettia pinnata).

BACKGROUND: Pongamia (Millettia pinnata syn. Pongamia pinnata), an oilseed legume species, is emerging as potential feedstock for sustainable biodiesel production. Breeding Pongamia for favorable traits in commercial application will rely on a comprehensive understanding of molecular mechanism regulating oil accumulation during its seed development. To date, only limited genomic or transcript sequences are available for Pongamia, while a temporal transcriptome profiling of developing seeds is still lacking in this species. RESULTS: In this work, we conducted a time-series analysis of morphological and physiological characters, oil contents and compositions, as well as global gene expression profiles in developing Pongamia seeds. Firstly, three major developmental phases were characterized based on the combined evidences from embryonic shape, seed weight, seed moisture content, and seed color. Then, the gene expression levels at these three phases were quantified by RNA-Seq analyses with three biological replicates from each phase. Nearly 94% of unigenes were expressed at all three phases, whereas only less than 2% of unigenes were exclusively expressed at one of these phases. A total of 8881 differentially expressed genes (DEGs) were identified between phases. Furthermore, the qRT-PCR analyses for 10 DEGs involved in lipid metabolism demonstrated a good reliability of our RNA-Seq data in temporal gene expression profiling. We observed a dramatic increase in seed oil content from the embryogenesis phase to the early seed-filling phase, followed by a steady and moderate increase towards the maximum at the desiccation phase. We proposed that a highly active expression of most genes related to fatty acid (FA) and triacylglycerol (TAG) biosynthesis at the embryogenesis phase might trigger both the substantial oil accumulation and the membrane lipid synthesis for rapid cell proliferation at this phase, while a concerted reactivation of TAG synthesis-related genes at the desiccation phase might further promote storage lipid synthesis to achieve the maximum content of seed oils. CONCLUSIONS: This study not only built a bridge between gene expression profiles and oil accumulation in developing seeds, but also laid a foundation for future attempts on genetic engineering of Pongamia varieties to acquire higher oil yield or improved oil properties for biofuel applications.

Virus-induced gene silencing (VIGS)-mediated functional characterization of two genes involved in lignocellulosic secondary cell wall formation.

KEY MESSAGE: Functional characterization of two tobacco genes, one involved in xylan synthesis and the other, a positive regulator of secondary cell wall formation, is reported. Lignocellulosic secondary cell walls (SCW) provide essential plant materials for the production of second-generation bioethanol. Therefore, thorough understanding of the process of SCW formation in plants is beneficial for efficient bioethanol production. Recently, we provided the first proof-of-concept for using virus-induced gene silencing (VIGS) approach for rapid functional characterization of nine genes involved in cellulose, hemicellulose and lignin synthesis during SCW formation. Here, we report VIGS-mediated functional characterization of two tobacco genes involved in SCW formation. Stems of VIGS plants silenced for both selected genes showed increased amount of xylem formation but thinner cell walls than controls. These results were further confirmed by production of stable transgenic tobacco plants manipulated in expression of these genes. Stems of stable transgenic tobacco plants silenced for these two genes showed increased xylem proliferation with thinner walls, whereas transgenic tobacco plants overexpressing these two genes showed increased fiber cell wall thickness but no change in xylem proliferation. These two selected genes were later identified as possible members of DUF579 family involved in xylan synthesis and KNAT7 transcription factor family involved in positive regulation of SCW formation, respectively. Glycome analyses of cell walls showed increased polysaccharide extractability in 1 M KOH extracts of both VIGS-NbDUF579 and VIGS-NbKNAT7 lines suggestive of cell wall loosening. Also, VIGS-NbDUF579 and VIGS-NbKNAT7 lines showed increased saccharification rates (74.5 and 40 % higher than controls, respectively). All these properties are highly desirable for producing higher quantities of bioethanol from lignocellulosic materials of bioenergy plants.

Enhanced accumulation of fatty acids and triacylglycerols in transgenic tobacco stems for enhanced bioenergy production.

KEY MESSAGE: We report a novel approach for enhanced accumulation of fatty acids and triacylglycerols for utilization as biodiesel in transgenic tobacco stems through xylem-specific expression of Arabidopsis DGAT1 and LEC2 genes. The use of plant biomass for production of bioethanol and biodiesel has an enormous potential to revolutionize the global bioenergy outlook. Several studies have recently been initiated to genetically engineer oil production in seeds of crop plants to improve biodiesel production. However, the "food versus fuel" issues have also sparked some studies for enhanced accumulation of oils in vegetative tissues like leaves. But in the case of bioenergy crops, use of woody stems is more practical than leaves. Here, we report the enhanced accumulation of fatty acids (FAs) and triacylglycerols (TAGs) in stems of transgenic tobacco plants expressing Arabidopsis diacylglycerol acyltransferase 1 (DGAT1) and leafy cotyledon2 (LEC2) genes under a developing xylem-specific cellulose synthase promoter from aspen trees. The transgenic tobacco plants accumulated significantly higher amounts of FAs in their stems. On an average, DGAT1 and LEC2 overexpression showed a 63 and 80% increase in total FA production in mature stems of transgenic plants over that of controls, respectively. In addition, selected DGAT1 and LEC2 overexpression lines showed enhanced levels of TAGs in stems with higher accumulation of 16:0, 18:2 and 18:3 TAGs. In LEC2 lines, the relative mRNA levels of the downstream genes encoding plastidic proteins involved in FA synthesis and accumulation were also elevated. Thus, here, we provide a proof of concept for our approach of enhancing total energy yield per plant through accumulation of higher levels of FAs in transgenic stems for biodiesel production.

Improved recombinant cellulase expression in chloroplast of tobacco through promoter engineering and 5' amplification promoting sequence.

Economical production of bioethanol from lignocellulosic biomass still faces many technical limitations. Cost-effective production of fermentable sugars is still not practical for large-scale production of bioethanol due to high costs of lignocellulolytic enzymes. Therefore, plant molecular farming, where plants are used as bioreactors, was developed for the mass production of cell wall degrading enzymes that will help reduce costs. Subcellular targeting is also potentially more suitable for the accumulation of recombinant cellulases. Herein, we generated transgenic tobacco plants (Nicotiana tabacum cv. SR1) that accumulated Thermotoga maritima BglB cellulase, which was driven by the alfalfa RbcsK-1A promoter and contained a small subunit of the rubisco complex transit peptide. The generated transformants possessed high specific BglB activity and did not show any abnormal phenotypes. Furthermore, we genetically engineered the RbcsK-1A promoter (MRbcsK-1A) and fused the amplification promoting sequence (aps) to MRbcsK-1A promoter to obtain high expression of BglB in transgenic plants. AMRsB plant lines with aps-MRbcsK-1A promoter showed the highest specific activity of BglB, and the accumulated BglB protein represented up to 9.3 % of total soluble protein. When BglB was expressed in Arabidopsis and tobacco plants, the maximal production capacity of recombinant BglB was 0.59 and 1.42 mg/g wet weight, respectively. These results suggests that suitable recombinant expression of cellulases in subcellular compartments such as chloroplasts will contribute to the cost-effective production of enzymes, and will serve as the solid foundation for the future commercialization of bioethanol production via plant molecular farming.

Importance of two consecutive methionines at the N-terminus of a cellulose synthase (PtdCesA8A) for normal wood cellulose synthesis in aspen.

All known orthologs of a secondary wall-associated cellulose synthase (CesA) gene from Arabidopsis, AtCesA8, encode CesA proteins with two consecutive methionines at their N-termini (MM or 2M). Here, we report that these 2Ms in an aspen ortholog of AtCesA8, PtdCesA8A, are important for maintaining normal wood cellulose biosynthesis in aspen trees. Overexpression of an altered PtdCesA8A cDNA encoding a PtdCesA8A protein missing one methionine at the N-terminus (1M) in aspen resulted in substantial decrease in cellulose content and caused negative effects on wood strength, suggesting that both methionines are essential for proper CesA expression and function in developing xylem tissues. Transcripts from a pair of paralogous native PtdCesA8 genes, as well as introduced PtdCesA8A:1M transgenes were significantly reduced in developing xylem tissues of transgenic aspen plants, suggestive of a co-suppression event. Overexpression of a native PtdCesA8A cDNA encoding a CesA protein with 2Ms at the N-terminus did not cause any such phenotypic changes. These results suggest the importance of 2Ms present at the N-terminus of PtdCesA8A protein during cellulose synthesis in aspen.

The transgenic poplar as an efficient bioreactor system for the production of xylanase.

Plants are attractive expression systems for large-scale, low-cost production of high-value proteins. The xylanase 2 gene (Xyn2), encoding an endo-ß-1,4-xylanase from Trichoderma reesei, was cloned and expressed in Escherichia coli and the poplar (Populus spp.). The optimal temperature and pH of the recombinant xylanase were 50 °C and 5.0 respectively when expressed in E. coli. The purpose of this study was to produce recombinant xylanase in poplar. The Xyn2 gene was transferred into poplars by Agrobacterium-mediated transformation. The transgenic status and transgene expression of the transformed poplar were confirmed by polymerase chain reaction (PCR) genotyping and reverse transcription (RT)-PCR analysis. The poplar-expressed xylanase was biologically active, with an expression level of up to 14.4% of total leaf soluble protein. In the leaves, the average xylanase content was 1.016 mg per g of leaf fresh weight in the transgenic poplar. We found that the poplar might make possible the large-scale production of commercially important recombinant proteins.

Perturbation of wood cellulose synthesis causes pleiotropic effects in transgenic aspen.

Genetic manipulation of cellulose biosynthesis in trees may provide novel insights into the growth and development of trees. To explore this possibility, the overexpression of an aspen secondary wall-associated cellulose synthase (PtdCesA8) gene was attempted in transgenic aspen (Populus tremuloides L.) and unexpectedly resulted in silencing of the transgene as well as its endogenous counterparts. The main axis of the transgenic aspen plants quickly stopped growing, and weak branches adopted a weeping growth habit. Furthermore, transgenic plants initially developed smaller leaves and a less extensive root system. Secondary xylem (wood) of transgenic aspen plants contained as little as 10% cellulose normalized to dry weight compared to 41% cellulose typically found in normal aspen wood. This massive reduction in cellulose was accompanied by proportional increases in lignin (35%) and non-cellulosic polysaccharides (55%) compared to the 22% lignin and 36% non-cellulosic polysaccharides in control plants. The transgenic stems produced typical collapsed or 'irregular' xylem vessels that had altered secondary wall morphology and contained greatly reduced amounts of crystalline cellulose. These results demonstrate the fundamental role of secondary wall cellulose within the secondary xylem in maintaining the strength and structural integrity required to establish the vertical growth habit in trees.

Virus-induced gene silencing offers a functional genomics platform for studying plant cell wall formation.

Virus-induced gene silencing (VIGS) is a powerful genetic tool for rapid assessment of plant gene functions in the post-genomic era. Here, we successfully implemented a Tobacco Rattle Virus (TRV)-based VIGS system to study functions of genes involved in either primary or secondary cell wall formation in Nicotiana benthamiana plants. A 3-week post-VIGS time frame is sufficient to observe phenotypic alterations in the anatomical structure of stems and chemical composition of the primary and secondary cell walls. We used cell wall glycan-directed monoclonal antibodies to demonstrate that alteration of cell wall polymer synthesis during the secondary growth phase of VIGS plants has profound effects on the extractability of components from woody stem cell walls. Therefore, TRV-based VIGS together with cell wall component profiling methods provide a high-throughput gene discovery platform for studying plant cell wall formation from a bioenergy perspective.

An update on the nomenclature for the cellulose synthase genes in Populus.

Cellulose synthase (CesA) is a central catalyst in the generation of the plant cell wall biomass and is, therefore, the focus of intense research. Characterization of individual CesA genes from Populus species has led to the publication of several different naming conventions for CesA gene family members in this model tree. To help reduce the resulting confusion, we propose here a new phylogeny-based CesA nomenclature that aligns the Populus CesA gene family with the established Arabidopsis thaliana CesA family structure.

Differential expression of three eucalyptus secondary cell wall-related cellulose synthase genes in response to tension stress.

Trees constitute the majority of lignocellulosic biomass existing on our planet. Trees also serve as important feedstock materials for various industrial products. However, little is known about the regulatory mechanisms of cellulose synthase (CesA) genes of trees. Here, the cloning and characterization of three CesA genes (EgraCesA1, EgraCesA2, and EgraCesA3) from an economically important tree species, Eucalyptus grandis, are reported. All three genes were specifically expressed in xylem cells of eucalyptus undergoing secondary cell wall biosynthesis. The GUS gene, expressed under the control of the EgraCesA2 or EgraCesA3 promoter, was also localized in the secondary xylem in transgenic tobacco stems. However, the EgraCesA1 promoter alone or along with its 5'-UTR introns was insufficient to direct appropriate GUS expression. EgraCesA2 and EgraCesA3 gene expression was up-regulated in tension-stressed eucalyptus xylem cells. Accordingly, GUS expression directed by the EgraCesA2 or EgraCesA3 promoter was also up-regulated. EgraCesA1 had no such response. Thus, it is most unlikely that EgraCesA1 is a subunit of the EgraCesA2-EgraCesA3 complex. The presence of at least two types of cellulose biosynthesis machinery in wood formation is an important clue in deciphering the underpinnings of the perennial growth of trees in various environmental conditions. By analysing GUS gene expression directed by the EgraCesA3 promoter or its deletions, several negative and positive regulatory regions controlling gene expression in xylem or phloem were identified. Also a region which is likely to contain mechanical stress-responsive elements was deduced. These results will guide further studies on identifying cis-regulatory elements directing CesA gene transcription and wood formation regulatory networks.

The cellulose paradox--simple molecule, complex biosynthesis.

Cellulose is the most abundant biopolymer on earth. Despite its simple structure, omnipresence in the plant kingdom, and ever increasing global importance as industrial raw material, the genetic and biochemical regulation of cellulose biosynthesis continues to be unclear. Over the past ten years, the advances in functional genomics have significantly improved our understanding of the processes of cellulose biosynthesis in higher plants. However, for each question answered myriad new unanswered ones have arisen.

Xylem-specific and tension stress-responsive coexpression of KORRIGAN endoglucanase and three secondary wall-associated cellulose synthase genes in aspen trees.

In nature, angiosperm trees develop tension wood on the upper side of their leaning trunks and drooping branches. Development of tension wood is one of the straightening mechanisms by which trees counteract leaning or bending of stem and resume upward growth. Tension wood is characterized by the development of a highly crystalline cellulose-enriched gelatinous layer next to the lumen of the tension wood fibers. Thus experimental induction of tension wood provides a system to understand the process of cellulose biosynthesis in trees. Since KORRIGAN endoglucanases (KOR) appear to play an important role in cellulose biosynthesis in Arabidopsis, we cloned PtrKOR, a full-length KOR cDNA from aspen xylem. Using RT-PCR, in situ hybridization, and tissue-print assays, we show that PtrKOR gene expression is significantly elevated on the upper side of the bent aspen stem in response to tension stress while KOR expression is significantly suppressed on the opposite side experiencing compression stress. Moreover, three previously reported aspen cellulose synthase genes, namely, PtrCesA1, PtrCesA2, and PtrCesA3 that are closely associated with secondary cell wall development in the xylem cells exhibited similar tension stress-responsive behavior. Our results suggest that coexpression of these four proteins is important for the biosynthesis of highly crystalline cellulose typically present in tension wood fibers. Their simultaneous genetic manipulation may lead to industrially relevant improvement of cellulose in transgenic crops and trees.

Differential expression patterns of two cellulose synthase genes are associated with primary and secondary cell wall development in aspen trees.

The quality and quantity of cellulose deposited in the primary and secondary cell walls of plants vary in accordance with their biological function. However, the molecular basis of such cellulose heterogeneity has so far remained unclear. Since enrichment of better-quality cellulose, in terms of increased degree of polymerization and crystallinity, is one of the goals of forest biotechnology, our main objective is to decipher the roles of distinct cellulose synthase (CesA) genes in tree development, with special reference to wood production. Here, we report two full-length CesA cDNAs, PtrCesA3 and PtrCesA4, from an economically important tree aspen (Populus tremuloides). PtrCesA3 is orthologous to the Arabidopsis AtCesA4 gene involved in secondary wall formation, whereas PtrCesA4 is orthologous to the Arabidopsis AtCesA1 gene involved in primary cell wall formation. To define the specific cell types expressing these CesA genes, we explored the natural distribution patterns of PtrCesA3 and PtrCesA4 transcripts in a variety of aspen organs, such as leaves, petiole, stem, and roots, using in situ hybridization with hypervariable region-specific antisense riboprobes. Such a side-by-side comparison suggested that PtrCesA3 is exclusively expressed in secondary-wall-forming cells of xylem and phloem fibers, whereas PtrCesA4 is predominantly expressed in primary-wall-forming expanding cells in all aspen organs examined. These findings suggest a functionally distinct role for each of these two types of PtrCesAs during primary and secondary wall biogenesis in aspen trees, and that such functional distinction appears to be conserved between annual herbaceous plants and perennial trees.

Differential expression patterns of two new primary cell wall-related cellulose synthase cDNAs, PtrCesA6 and PtrCesA7 from aspen trees.

Based on elegant molecular genetic analyses, distinct classes of cellulose synthase (CesA) genes have been associated with either primary or secondary cell wall development in Arabidopsis. Here, we report on cloning of two new CesA cDNAs, PtrCesA6 and PtrCesA7 involved in the primary cell wall development in aspen (Populus tremuloides) trees. Both these distinct cDNAs, isolated from a developing xylem cDNA library, share only 60-67% identities with each other as well as with five other previously known aspen CesA cDNAs. Interestingly, PtrCESA6 from aspen, a dicot species, shares maximum identity of 81-84% with three CESA isoforms from maize and rice, two monocot species. On the other hand, PtrCESA7 shares a maximum identity of 86% with AtCESA2, a primary wall-related CesA member from Arabidopsis, a dicot species. Gene expression analyses by reverse transcriptase-polymerase chain reactions (RT-PCRs) suggested that both these genes are expressed at a low level in all aspen tissues examined but PtrCesA7 is expressed at a higher level than PtrCesA6. While corroborating these results, in situ mRNA hybridization studies using three different aspen organs also suggested that PtrCesA6 and PtrCesA7 genes are expressed in all expanding cells depositing primary cell wall but PtrCesA7 is expressed at a higher level than PtrCesA6. These differential gene expression profiles suggest that each of these CesAs may be playing a specific role during primary cell wall development in aspen trees. Isolation of two primary wall related CesA genes from xylem tissues also suggest their importance during xylem development, which is traditionally considered to be enriched in secondary cell wall forming cells of economical significance.

Suppression subtractive hybridization-mediated transcriptome analysis from multiple tissues of aspen (Populus tremuloides) altered in phenylpropanoid metabolism.

A PCR-based suppression subtractive hybridization (SSH) technique was used to identify differentially expressed genes in developing tissues of control and transgenic aspen (Populus tremuloides Michx.) with down-regulated 4CL1 (4-coumarate:coenzyme A ligase) expression and enhanced growth. A total of 11,308 expressed sequence tags (ESTs) representing 5,028 non-redundant transcripts encoding 4,224 unique proteins was obtained from shoot apex, young stem, young leaf and root tip SSH libraries. Putative functions can be assigned to 60% of these transcripts. Approximately 14% of the ESTs are not represented among the 111,000 entries already present in Populus EST databases. In general, ESTs of the metabolism class occurred at a higher frequency in control- than transgenic-enriched libraries of all tissues, whereas protein synthesis and protein fate ESTs were over-represented in meristematic tissues of transgenics where 4CL1 was relatively strongly suppressed. Among all tissues, leaves yielded the highest percentage of ESTs with either unknown protein function or insignificant similarity to other protein/DNA/EST sequences in existing databases. Of particular interest was a large number of ESTs (16%) associated with signal transduction in transgenic leaves. Among these were several leucine-rich-repeat receptor-like protein kinases with markedly elevated expression in transgenic leaves. We also identified homologs of transposable elements that were up-regulated in transgenic tissues, providing the first experimental data for active expression of DNA mobile elements in long-lived tree species.

Cloning and characterization of cellulose synthase-like gene, PtrCSLD2 from developing xylem of aspen trees.

Genetic improvement of cell wall polymer synthesis in forest trees is one of the major goals of forest biotechnology that could possibly impact their end product utilization. Identification of genes involved in cell wall polymer biogenesis is essential for achieving this goal. Among various candidate cell wall-related genes, cellulose synthase-like D (CSLD) genes are intriguing due to their hitherto unknown functions in cell wall polymer synthesis but strong structural similarity with cellulose synthases (CesAs) involved in cellulose deposition. Little is known about CSLD genes from trees. In the present article PtrCSLD2, a first CSLD gene from an economically important tree, aspen (Populus tremuloides) is reported. PtrCSLD2 cDNA was isolated from an aspen xylem cDNA library and encodes a protein that shares 90% similarity with Arabidopsis AtCSLD3 protein involved in root hair tip growth. It is possible that xylem fibers that also grow by intrusive tip growth may need expression of PtrCSLD2 for controlling the length of xylem fibers, a wood quality trait of great economical importance. PtrCSLD2 protein has a N-terminal cysteine-rich putative zinc-binding domain; eight transmembrane domains; alternating conserved and hypervariable domains; and a processive glycosyltransferases signature, D, D, D, QXXRW; all similar to aspen CesA proteins. However, PtrCSLD2 shares only 43-48% overall identity with the known aspen CesAs suggesting its distinct functional role in cell wall polymer synthesis perhaps other than cellulose biosynthesis. Based on Southern analysis, the aspen CSLD gene family consists of at least three genes and this gene copy estimate is supported by phylogenetic analysis of available CSLDs from plants. Moreover, gene expression studies using RT-PCR and in situ mRNA hybridization showed that PtrCSLD2 is expressed at a low level in all aspen tissues examined with a slightly higher expression level in secondary cell wall-enriched aspen xylem as compared to primary cell wall enriched tissues. Together, these observations suggest that PtrCSLD2 gene may be involved in the synthesis of matrix polysaccharides that are dominant in secondary cell walls of poplar xylem. Future molecular genetic analyses will clarify the functional significance of CSLD genes in the development of woody trees.