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.     

A new cellulose synthase gene (PtrCesA2) from aspen xylem is orthologous to Arabidopsis AtCesA7 (irx3) gene associated with secondary cell wall synthesis

We report here the molecular cloning and characterization of a new full-length cellulose synthase (CesA) cDNA, PtrCesA2 from aspen (Populus tremuloides) trees. The predicted PtrCesA2 protein shows a high degree of identity/similarity (87%/91%) to the predicted gene product of Arabidopsis AtCesA7 gene that has been associated with secondary cell wall development. Previously, a mutation in AtCesA7 gene (irx3) was correlated with a significant decrease in the amount of cellulose synthesized (about 70%) and genetic complementation of irx3 mutant with a wild-type AtCesA7 gene restored the normal phenotype. This is the first report of a full-length AtCesA7 ortholog from any non-Arabidopsis species. Interestingly, PtrCesA2 shares only 64% identity with our earlier reported PtrCesA1 from aspen suggesting its structural distinctness from the only other known CesA member from the aspen genome. PtrCesA1 is a xylem-specific and tension stress responsive gene that is highly similar to another Arabidopsis gene, AtCesA8 which also has been associated with secondary wall development. Moreover, AtCesA7 and AtCesA8 are suggested to be part of the same cellulose synthase complex. Isolation of PtrCesA2 from a xylem library enriched in cells with active secondary wall synthesis, PtrCesA2 expression levels similar to PtrCesA1 and high similarity of PtrCesA1 and PtrCesA2 to AtCesA8 and AtCesA7, respectively, suggest that both these aspen genes might be involved in the secondary wall development in aspen woody tissues. Availability of two aspen CesA orthologs will now enable us to examine if PtrCesA1 and PtrCesA2 functionally interact during aspen wood development that has long-term implications on genetic improvement of forest trees.     

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.Suchita Bhandari and Takeshi Fujino contributed equally to this research.     

The seasonal activity and the effect of mechanical bending and wounding on the PtCOMT promoter in Betula pendula Roth

In this study, 900-bp (signed as p including nucleotides –1 to –886) and partly deleted (signed as dp including nucleotides –1 to –414) COMT (caffeate/5-hydroxyferulate O-methyltransferase) promoters from Populus tremuloides Michx. were fused to the GUS reporter gene, and the tissue-specific expression patterns of the promoters were determined in Betula pendula Roth along the growing season, and as a response to mechanical bending and wounding. The main activity of the PtCOMTp- and PtCOMTdp-promoters, determined by the histochemical GUS assay, was found in the developing xylem of stems during the 8th–13th week and in the developing xylem of roots in the 13th week of the growing season. The GUS expression patterns did not differ among the xylem cell types. The PtCOMT promoter-induced GUS expression observed in phloem fibres suggests a need for PtCOMT expression and thus syringyl (S) lignin synthesis in fibre lignification. However, the PtCOMTdp-promoter induced GUS expression in stem trichomes, which may contribute to the biosynthesis of phenylpropanoid pathway-derived compounds other than lignin. Finally, a strong GUS expression was induced by the PtCOMT promoters in response to mechanical stem bending but not to wounding. The lack of major differences between the PtCOMTp- and PtCOMTdp-promoters suggests that the deleted promoter sequence (including nucleotides −415 to −886) did not contain a significant regulatory element contributing to the GUS expression in young B. pendula trees.     

Three loblolly pine CesA genes expressed in developing xylem are orthologous to secondary cell wall CesA genes of angiosperms

Specific plant cellulose synthases (CesA), encoded by a multigene family, are necessary for secondary wall synthesis in vascular tissues and are critical to wood production. We obtained full-length clones for the three CesAs that are highly expressed in developing xylem and examined their phylogenetic relationships and expression patterns in loblolly pine tissues. Full-length CesA clones were isolated from cDNA of developing loblolly pine (Pinus taeda) xylem and phylogenetic inferences made from plant CesA protein sequences. Expression of the three genes was examined by Northern blot analysis and semiquantitative RT-PCR. Each of three PtCesA genes is orthologous to one of the three angiosperm secondary cell wall CesAs. The PtCesAs are coexpressed in tissues of loblolly pine with tissues undergoing secondary cell wall biosynthesis showing the highest levels of expression. Phylogenetic and expression analyses suggest that functional roles for these loblolly pine CesAs are analogous to those of orthologs in angiosperm taxa. Based upon evidence from this and other studies, we suggest division of seed plant CesA genes into six major paralogous groups, each containing orthologs from various taxa. Available evidence suggests that paralogous CesA genes and their distinct functional roles evolved before the divergence of gymnosperm and angiosperm lineages.     

Down-regulation of an anionic peroxidase in transgenic aspen and its effect on lignin characteristics

It is generally accepted that peroxidases catalyze the final step in the biosynthesis of lignin. In this study, to examine how expression of prxA3a, a gene for an anionic peroxidase, might be related to lignification in plant tissues, we produced transgenic tobacco plants that harbored a gene for β-glucuronidase (GUS) fused to the prxA3a promoter. Histochemical staining for GUS activity indicated that the prxA3a promoter was active mainly in the lignifying cells of stem tissues. Further, to examine the effects of suppressing the expression of prxA3a, we transferred an antisense prxA3a gene construct into the original host, hybrid aspen (Populus sieboldii ×P. gradidentata), under the control of the original promoter of the prxA3a gene. Eleven transformed aspens were obtained and characterized, and the stable integration of the antisense construct was confirmed by PCR and Southern blotting analysis in all these lines. Assays of enzymatic activity showed that both total peroxidase activity and acidic peroxidase activity were lower in most transgenic lines than in the control plants. In addition, the reduction of peroxidase activity was associated with lower lignin content and modified lignin composition. Transgenic lines with the highest reduction of peroxidase activity displayed a higher syringyl/vanillin (S/V) ratio and a lower S+V yield, mainly because of a decreased amount of V units. Thus, our results indicate that prxA3a is involved in the lignification of xylem tissue and that the down-regulation of anionic peroxidase alters both lignin content and composition in hybrid aspen.     

Cambial-region-specific expression of the Agrobacterium iaa genes in transgenic aspen visualized by a linked uidA reporter gene

The level of indole-3-acetic acid (IAA) was locally modified in cambial tissues of transgenic aspen (Populus tremula L. x Populus tremuloides Michx.). We also demonstrate the use of a linked reporter gene to visualize the expression of the iaa genes. The rate-limiting bacterial IAA-biosynthetic gene iaaM and the reporter gene for beta-glucuronidase (GUS), uidA, were each fused to the cambial-region-specific Agrobacterium rhizogenes rolC promoter and linked on the same T-DNA. In situ hybridization of the iaaM gene confirmed that histochemical analysis of GUS activity could be used to predict iaaM gene expression. Moreover, quantitative fluorometric analysis of GUS activity allowed estimation of the level of de novo production of IAA in transgenic lines carrying a single-copy insert of the iaaM, uidA T-DNA. Microscale analysis of the IAA concentration across the cambial region tissues showed an increase in IAA concentration of about 35% to 40% in the two transgenic lines, but no changes in the radial distribution pattern of IAA compared with wild-type plants. This increase did not result in any changes in the developmental pattern of cambial derivatives or the cambial growth rate, which emphasizes the importance of the radial distribution pattern of IAA in controlling the development of secondary xylem, and suggests that a moderate increase in IAA concentration does not necessarily stimulate growth.     

Repression of lignin biosynthesis promotes cellulose accumulation and growth in transgenic trees.

Because lignin limits the use of wood for fiber, chemical, and energy production, strategies for its downregulation are of considerable interest. We have produced transgenic aspen (Populus tremuloides Michx.) trees in which expression of a lignin biosynthetic pathway gene Pt4CL1 encoding 4-coumarate:coenzyme A ligase (4CL) has been downregulated by antisense inhibition. Trees with suppressed Pt4CL1 expression exhibited up to a 45% reduction of lignin, but this was compensated for by a 15% increase in cellulose. As a result, the total lignin-cellulose mass remained essentially unchanged. Leaf, root, and stem growth were substantially enhanced, and structural integrity was maintained both at the cellular and whole-plant levels in the transgenic lines. Our results indicate that lignin and cellulose deposition could be regulated in a compensatory fashion, which may contribute to metabolic flexibility and a growth advantage to sustain the long-term structural integrity of woody perennials.     

Influence of over-expression of the FLOWERING PROMOTING FACTOR 1 gene (FPF1) from Arabidopsis on wood formation in hybrid poplar (Populus tremula L. × P. tremuloides Michx.)

Constitutive expression of the FPF1 gene in hybrid aspen (Populus tremula L. × P. tremuloides Michx.) showed a strong effect on wood formation but no effect on flowering time. Gene expression studies showed that activity of flowering time genes PtFT1, PtCO2, and PtFUL was not increased in FPF1 transgenic plants. However, the SOC1/TM3 class gene PTM5, which has been related to wood formation and flowering time, showed a strong activity in stems of all transgenic lines studied. Wood density was lower in transgenic plants, despite significantly reduced vessel frequency which was overcompensated by thinner fibre cell walls. Chemical screening of the wood by pyrolysis GC/MS showed that FPF1 transgenics have higher fractions of cellulose and glucomannan products as well as lower lignin content. The latter observation was confirmed by UV microspectrophotometry on a cellular level. Topochemical lignin distribution revealed a slower increase of lignin incorporation in the developing xylem of the transgenics when compared with the wild-type plants. In line with the reduced wood density, micromechanical wood properties such as stiffness and ultimate stress were also significantly reduced in all transgenic lines. Thus, we provide evidence that FPF1 class genes may play a regulatory role in both wood formation and flowering in poplar.     

Gene expression profiles in different stem internodes reveal the genetic regulation of primary and secondary stem development in <Emphasis Type="Italic">Betula platyphylla</Emphasis>

Secondary growth of stems is an important process for the radial increase of trees. To gain an insight into the molecular mechanisms underlying stem development from primary to secondary growth and to provide information for molecular research and breeding in Betula platyphylla (birch), the gene expression profiles of material from the first, third, and fifth internodes (IN) of 3-month-old seedlings were analyzed. Compared with the first IN, 177 genes were up-regulated and 157 genes down-regulated in the third IN; in the fifth IN, 180 genes were up-regulated and 275 genes were down-regulated. The expressions of 24 genes were up-regulated and 6 genes were down-regulated in the fifth IN relative to the third IN. The differentially expressed genes were annotated as having roles in cambium, xylem, and phloem development and formation; including cell wall expansion, cellulose biosynthesis, lignin biosynthesis and deposition, xylem extension, cell wall modification, and growth hormone responses. The expressions of genes related to cell wall expansion and cellulose biosynthesis in the primary cell wall were down-regulated in the third and fifth IN relative to the first IN. Genes involved in lignin biosynthesis, xylem extension, and cellulose synthesis in the secondary cell wall were up-regulated in the third and fifth IN relative to the first IN. These results described the patterns of gene expression during stem development in birch and provided candidate genes for further functional characterization.     

Downregulation of high-isoelectric-point extracellular superoxide dismutase mediates alterations in the metabolism of reactive oxygen species and developmental disturbances in hybrid aspen

Transgenic hybrid aspen (Populus tremula L. x P. tremuloides Michx.) plants expressing a high-isoelectric-point superoxide dismutase (hipI-SOD) gene in antisense orientation were generated to investigate its function. Immunolocalization studies showed the enzyme to be localized extracellularly, in the secondary cell wall of xylem vessels and phloem fibers. The antisense lines of hipI-SOD exhibited a distinct phenotype; growth rate was reduced, stems were thinner and leaves smaller than in wild-type (WT) plants. The abundance of hipI-SOD was reduced in the bark and xylem of plants from these antisense lines. The vascular tissue of transgenic lines became lignified earlier than in WT plants and also showed an increased accumulation of reactive oxygen species (ROS). Xylem fibers and vessels were shorter and thinner in the transgenic lines than in WT plants. The total phenolic content was enhanced in the antisense lines. Furthermore, microarray analysis indicated that several enzymes involved in cell signaling, lignin biosynthesis and stress responses were upregulated in apical vascular tissues of transgenic plants. The upregulation of selected genes involved in lignin biosynthesis was also verified by real-time PCR. The results suggest that, in the transgenic plants, a premature transition into maturation occurs and the process is discussed in terms of the effects of increased accumulation of ROS due to reduced expression of hipI-SOD during development and differentiation.     

Metabolic engineering of p-hydroxybenzoate in poplar lignin

Ester-linked p-hydroxybenzoate occurs naturally in poplar lignin as pendent groups that can be released by mild alkaline hydrolysis. These ‘clip-off’ phenolics can be separated from biomass and upgraded into diverse high-value bioproducts. We introduced a bacterial chorismate pyruvate lyase gene into transgenic poplar trees with the aim of producing more p-hydroxybenzoate from chorismate, itself a metabolic precursor to lignin. By driving heterologous expression specifically in the plastids of cells undergoing secondary wall formation, this strategy achieved a 50% increase in cell-wall-bound p-hydroxybenzoate in mature wood and nearly 10 times more in developing xylem relative to control trees. Comparable amounts also remained as soluble p-hydroxybenzoate-containing xylem metabolites, pointing to even greater engineering potential. Mass spectrometry imaging showed that the elevated p-hydroxybenzoylation was largely restricted to the cell walls of fibres. Finally, transgenic lines outperformed control trees in assays of saccharification potential. This study highlights the biotech potential of cell-wall-bound phenolate esters and demonstrates the importance of substrate supply in lignin engineering.     

Expression of a glycine decarboxylase complex H-protein in non-photosynthetic tissues of Populus tremuloides

The Gly decarboxylase complex (GDC) is abundant in mitochondria of C3 leaves and functions in photorespiratory carbon recovery. However, expression of GDC component proteins has generally been less evident in non-green tissues. Here we report an aspen (Populus tremuloides Michx.) PtgdcH1 gene, encoding a GDC subunit H-protein that is phylogenetically distinct from previously characterized photorespiratory H-proteins. Strong expression of PtgdcH1 in root tips and developing xylem suggests that GDC supports a very active C1 metabolism in non-photosynthetic tissues of aspen.     

The Last Step of Syringyl Monolignol Biosynthesis in Angiosperms Is Regulated by a Novel Gene Encoding Sinapyl Alcohol Dehydrogenase

Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) has been thought to mediate the reduction of both coniferaldehyde and sinapaldehyde into guaiacyl and syringyl monolignols in angiosperms. Here, we report the isolation of a novel aspen gene (PtSAD) encoding sinapyl alcohol dehydrogenase (SAD), which is phylogenetically distinct from aspen CAD (PtCAD). Liquid chromatography-mass spectrometry-based enzyme functional analysis and substrate level-controlled enzyme kinetics consistently demonstrated that PtSAD is sinapaldehyde specific and that PtCAD is coniferaldehyde specific. The enzymatic efficiency of PtSAD for sinapaldehyde was approximately 60 times greater than that of PtCAD. These data suggest that in addition to CAD, discrete SAD function is essential to the biosynthesis of syringyl monolignol in angiosperms. In aspen stem primary tissues, PtCAD was immunolocalized exclusively to xylem elements in which only guaiacyl lignin was deposited, whereas PtSAD was abundant in syringyl lignin-enriched phloem fiber cells. In the developing secondary stem xylem, PtCAD was most conspicuous in guaiacyl lignin-enriched vessels, but PtSAD was nearly absent from these elements and was conspicuous in fiber cells. In the context of additional protein immunolocalization and lignin histochemistry, these results suggest that the distinct CAD and SAD functions are linked spatiotemporally to the differential biosynthesis of guaiacyl and syringyl lignins in different cell types. SAD is required for the biosynthesis of syringyl lignin in angiosperms.