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.     

Differential expression of two spermidine synthase genes during early fruit development and in vegetative tissues of pea

Two cDNAs from young pea fruits coding for functional spermidine synthases (EC 2.5.1.16) were isolated. The corresponding genes were named psSPDSYN1 and psSPDSYN2. Both cDNAs complemented spe3 gene when introduced into the Y480 strain of Saccharomyces cerevisiae, which is a null mutant for the spermidine synthase gene. psSPDSYN1 and psSPDSYN2 are regulated differentially. psSPDSYN1 is up-regulated early after fruit set whereas psSPDSYN2 is expressed later. Spermidine synthase activity was detected in pea ovaries, and correlates with the pattern of expression of psSPDSYN1. In the pea plant, psSPDSYN1 is highly expressed in actively growing tissues, whereas the highest level of psSPDSYN2 mRNA was detected in fully elongated stem.     

Analysis of the cellulose synthase genes associated with primary cell wall synthesis in Bambusa oldhamii

The synthesis of cell wall polysaccharides is highly active in rapidly growing bamboo shoots. We cloned a set of BoCesA cDNAs that encode cellulose synthase from bamboo (Bambusa oldhamii) and investigated the expression patterns of the BoCesA2, BoCesA5, BoCesA6 and BoCesA7 genes. The four BoCesA genes were differentially expressed in the different parts of growing bamboo shoots, in various organs, and in multiple shoots that were cultured in vitro. They were down-regulated by α-naphthaleneacetic acid and differentially affected by thidiazuron in the multiple shoots. In situ RT-PCR analyses demonstrated that BoCesA2, BoCesA5, BoCesA6, and BoCesA7 mRNAs were present throughout the base and the internode regions of the etiolated shoots that emerged from pseudorhizomes, and in the internode regions of the juvenile branch shoots that emerged from nodes of mature bamboo culms; however, the expression of the four genes in the lignified internode of the branch shoot was predominantly detected in the center of the vascular bundles. Our results for cDNA cloning, expression analyses, and phylogenetic analysis suggest that the 10 BoCesA genes cloned from the etiolated bamboo shoots participate in cellulose synthesis in the primary cell walls of the growing bamboo, and that at least three additional BoCesA genes involved in cellulose synthesis in the secondary walls may be present in the bamboo genome. The expressions of BoCesA genes may be under fine control in response to the various developmental stages and physiological conditions of bamboo.     

Distribution of plasma membrane rosettes and kinetics of cellulose formation in xylem development of higher plants

Summary Germ roots of several higher plants—maize (Zea mays), mung bean (Vigna radiata) and cress (Lepidium sativum)—were freeze-fractured without cryoprotection in order to confirm and extend the informations on frequency and distribution of plasma membrane particle complexes with respect to cellulose formation. In all three objects the PF of developing xylem elements showed rosette accumulations in the regions of wall thickenings. The rosette-distribution pattern ranges from random in a young stage, to more grouped in a probable intermediate stage to strictly localized in later stages. The frequency of rosettes increases from stage to stage.In all three objects the EF of developing xylem elements is relatively poor in particles. Observations of terminal globules were rare and undistinct. This leads to the assumption that rosettes on the PF and terminal globules on the EF are not part of the same complex.A comparison of the number and distribution of microtubules underlying the xylem wall thickenings with rosette frequency and distribution leads to the conclusion that there seem to be no direct connections between these two structures. Microtubules may be involved in grouping of rosettes, thus indirectly orienting microfibril deposition. Calculations based on the observed rosette frequencies and the amount of wall material formed indicate that in xylem development 1,000 nm elementary fibril per rosette per minute may be formed and that the active phase of one rosette may be about 10 minutes.Abbreviations EF exoplasmic fracture face - PF protoplasmic fracture face     

Genetic evidence that the two isozymes of sucrose synthase present in developing maize endosperm are critical, one for cell wall integrity and the other for starch biosynthesis

In maize, two paralogous genes, Sh1 and Sus1, encode two biochemically similar isozymes of sucrose synthase, SS1 and SS2, respectively. Previous studies have attributed the mild starch deficiency of the shrunken1 (sh1) endosperm to the loss of the SS1 isozyme in the mutant. Here we describe the first mutation in the sucrose synthase1 (Sus1) gene, sus1-1, and the isolation of a double recessive genotype, sh1 sus1-1. Combined data from diverse studies, including Northern and Western analyses, RT-PCR and genomic PCR, cloning and sequencing data for the 3′ region, show that the mutant sus1-1 gene has a complex pattern of expression, albeit at much reduced levels as compared to the Sus1 gene. Endosperm sucrose synthase activity in sh1 sus1-1 was barely 0.5% of the total activity in the Sh1 Sus1 genotype. Significantly, comparative analyses of Sh1 Sus1, sh1 Sus1 and sh1 sus1-1 genotypes have, for the first time, allowed us to dissect the relative contributions of each isozyme to endosperm development. Starch contents in endosperm of the three related genotypes were 100, 78 and 53%, respectively. Anatomical analyses, which confirmed the previously described early cell degeneration phenotype unique to the sh1 Sus1 endosperm, revealed no detectable difference between the two sh1 genotypes. We conclude that the SS1 isozyme plays the dominant role in providing the substrate for cellulose biosynthesis, whereas the SS2 protein is needed mainly for generating precursors for starch biosynthesis. Received: 22 January 1998 / Accepted: 30 March 1998     

Cell-wall structure and anisotropy in procuste, a cellulose synthase mutant of Arabidopsis thaliana

In dark-grown hypocotyls of the Arabidopsis procuste mutant, a mutation in the CesA6 gene encoding a cellulose synthase reduces cellulose synthesis and severely inhibits elongation growth. Previous studies had left it uncertain why growth was inhibited, because cellulose synthesis was affected before, not during, the main phase of elongation. We characterised the quantity, structure and orientation of the cellulose remaining in the walls of affected cells. Solid-state NMR spectroscopy and infrared microscopy showed that the residual cellulose did not differ in structure from that of the wild type, but the cellulose content of the prc-1 cell walls was reduced by 28%. The total mass of cell-wall polymers per hypocotyl was reduced in prc-1 by about 20%. Therefore, the fourfold inhibition of elongation growth in prc-1 does not result from aberrant cellulose structure, nor from uniform reduction in the dimensions of the cell-wall network due to reduced cellulose or cell-wall mass. Cellulose orientation was quantified by two quantitative methods. First, the orientation of newly synthesised microfibrils was measured in field-emission scanning electron micrographs of the cytoplasmic face of the inner epidermal cell wall. The ordered transverse orientation of microfibrils at the inner face of the cell wall was severely disrupted in prc-1 hypocotyls, particularly in the early growth phase. Second, cellulose orientation distributions across the whole cell-wall thickness, measured by polarised infrared microscopy, were much broader. Analysis of the microfibril orientations according to the theory of composite materials showed that during the initial growth phase, their anisotropy at the plasma membrane was sufficient to explain the anisotropy of subsequent growth.     

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.     

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.     

Differential expression of two distinct phenylalanine ammonia-lyase genes in condensed tannin-accumulating and lignifying cells of quaking aspen

Lignins, along with condensed tannins (CTs) and salicylate-derived phenolic glycosides, constitute potentially large phenylpropanoid carbon sinks in tissues of quaking aspen (Populus tremuloides Michx.). Metabolic commitment to each of these sinks varies during development and adaptation, and depends on L-phenylalanine ammonia-lyase (PAL), an enzyme catalyzing the deamination of L-phenylalanine to initiate phenylpropanoid metabolism. In Populus spp., PAL is encoded by multiple genes whose expression has been associated with lignification in primary and secondary tissues. We now report cloning two differentially expressed PAL cDNAs that exhibit distinct spatial associations with CT and lignin biosynthesis in developing shoot and root tissues of aspen. PtPAL1 was expressed in certain CT-accumulating, non-lignifying cells of stems, leaves, and roots, and the pattern of PtPAL1 expression varied coordinately with that of CT accumulation along the primary to secondary growth transition in stems. PtPAL2 was expressed in heavily lignified structural cells of shoots, but was also expressed in non-lignifying cells of root tips. Evidence of a role for Pt4CL2, encoding 4-coumarate:coenzyme A ligase, in determining CT sink strength was gained from cellular co-expression analysis with PAL1 and CTs, and from experiments in which leaf wounding increased PAL1 and 4CL2 expression as well as the relative allocation of carbon to CT with respect to phenolic glycoside, the dominant phenolic sink in aspen leaves. Leaf wounding also increased PAL2 and lignin pathway gene expression, but to a smaller extent. The absence of PAL2 in most CT-accumulating cells provides in situ support for the idea that PAL isoforms function in specific metabolic milieus.     

Functional analysis of complexes with mixed primary and secondary cellulose synthases

In higher plants, cellulose is synthesized by cellulose synthase complexes, which contain multiple isoforms of cellulose synthases (CESAs). Among the total 10 CESA genes in Arabidopsis, recessive mutations at three of them cause the collapse of mature xylem cells in inflorescence stems of Arabidopsis (irx1^cesa8, irx3^cesa7 and irx5^cesa4). These CESA genes are considered secondary cell wall CESAs. The others (the function CESA10 is still unknown) are thought to be specialized for cellulose synthesis in the primary cell wall. A split-ubiquitin membrane yeast two-hybrid system was used to assess interactions among four primary CESAs (CESA1, CESA2, CESA3, CESA6) and three secondary CESAs (CESA4, CESA7, CESA8). Our results showed that primary CESAs could physically interact with secondary CESAs in a limited fashion. Analysis of transgenic lines showed that CESA1 could partially rescue irx1^cesa8 null mutants, resulting in complementation of the plant growth defect, collapsed xylem and cellulose content deficiency. These results suggest that mixed primary and secondary CESA complexes are functional using experimental set-ups.     

Lignin isolated from primary walls of hybrid aspen cell cultures indicates significant differences in lignin structure between primary and secondary cell wall.

Hybrid aspen (Populus tremula x tremuloides) cell cultures were grown for 7, 14 and 21 days. The cell cultures formed primary cell walls but no secondary cell wall according to carbohydrate analysis and microscopic characterization. The primary walls were lignified, increasingly with age, according to Klason lignin analysis. Presence of lignin in the primary walls, with a higher content in 21-day old cells than in 7-day old cells, was further supported by phloroglucinol/HCl reagent test and confocal microscopy after both immunolocalization and staining with acriflavin. Both laccase and peroxidase activity were found in the cultures and the activity increased during lignin formation. The lignin from the cell culture material was compared to lignin from mature aspen wood, where most of the lignin originates in the secondary cell wall, and which served as our secondary cell wall control. Lignin from the cell walls was isolated and characterized by thioacidolysis followed by gas chromatography and mass spectrometry. The lignin in the cell cultures differed from lignin of mature aspen wood in that it consisted exclusively of guaiacyl units, and had a more condensed structure. Five lignin structures were identified by mass spectrometry in the cell suspension cultures. The results indicate that the hybrid aspen cell culture used in this investigation may be a convenient experimental system for studies of primary cell wall lignin.     

Differential expression of two β-amylase genes of rye during seed development

The expression of two β-amylase loci was analysed in the developing seeds of two inbred lines of rye (Secale cereale L.), one of which was a β-amylase deficient mutant. Enzymatic activity and the contents of enzymatic protein and mRNA specific for each of an endosperm-characteristic and ubiquitous β-amylase were determined throughout the course of caryopsis development. Both loci were expressed in the developing normal line caryopses according to different temporal and quantitative patterns. The ubiquitous enzyme-specific locus β-Amy 2 was expressed earlier; both mRNA and enzymatic protein accumulated to a maximum extent at 10 to 15 days after pollination. In contrast, the highest content of mRNA for endosperm β-amylase (encoded by the β-Amy I locus) was found 20 days after pollination, and the corresponding enzymatic protein accumulated throughout seed development. The expression of the β-Amy I locus was 30- to 40-fold higher than that of the β-Amy 2 locus in terms of maximum specific mRNA accumulation. The expression product of only the β-Amy 2 locus was found in the developing mutant line caryopses. The expression pattern of this locus was similar in the developing normal and mutant line seeds in terms of the temporal accumulation of mRNA and enzymatic protein. However, an approximately 4-fold higher level of ubiquitous β-amylase-specific mRNA was found in the mutant than in the normal line caryopses, and the content of ubiquitous β-amylase protein decreased to near zero at seed maturity in the mutant line, but not in the normal line, caryopses. The enzymatic activities of both β-amylases appeared to be regulated at the level of accumulated enzymatic protein.     

Ectopic expression of SOD and APX genes in Arabidopsis alters metabolic pools and genes related to secondary cell wall cellulose biosynthesis and improve salt tolerance

Molecular Biology Reports - Hydrogen peroxide (H2O2) is known to accumulate in plants during abiotic stress conditions and also acts as a signalling molecule. In this study, Arabidopsis thaliana...     

Hydrogen peroxide and expression of hipI-superoxide dismutase are associated with the development of secondary cell walls in Zinnia elegans

A special form of a CuZn-superoxide dismutase with a high isoelectric point (hipI-SOD; EC 1.15.1.1) and hydrogen peroxide (H2O2) production were studied during the secondary cell wall formation of the inducible tracheary element cell-culture system of Zinnia elegans L. Confocal microscopy after labelling with 2',7'-dichlorofluorescin diacetate showed H2O2 to be located largely in the secondary cell walls in developing tracheary elements. Fluorescence-activated cell sorting analysis showed there were lower levels of H2O2 in the population containing tracheary elements when H2O2 scavengers such as ascorbate, catalase, and reduced glutathione were applied to the cell culture. Inhibitors of NADPH oxidase and SOD also reduced the amount of H2O2 in the tracheary elements. Furthermore, addition of these compounds to cell cultures at the time of tracheary element initiation reduced the amount of lignin and the development of the secondary cell walls. Analysis of UV excitation under a confocal laser scanning microscope confirmed these results. The expression of hipI-SOD increased as the number of tracheary elements in the cell culture increased and developed. Additionally, immunolocalization of a hipI-SOD isoform during the tracheary element differentiation showed a developmental build-up of the protein in the Golgi apparatus and the secondary cell wall. These findings suggest a novel hipI-SOD could be involved in the regulation of H2O2 required for the development of the secondary cell walls of tracheary elements.     

Interactions between membrane-bound cellulose synthases involved in the synthesis of the secondary cell wall

It has not yet been reported how the secondary CESA (cellulose synthase) proteins are organized in the rosette structure. A membrane-based yeast two-hybrid (MbYTH) approach was used to analyze the interactions between the CESA proteins involved in secondary cell wall synthesis of Arabidopsis and the findings were confirmed in planta by bimolecular fluorescence complementation (BiFC) assay. Results indicated that although all CESA proteins can interact with each other, only CESA4 is able to form homodimers. A model is proposed for the secondary rosette structure. The RING-motif proved not to be essential for the interaction between the CESA proteins.

Structured summary

MINT-6951243: PIP2-1 (uniprotkb:P43286) physically interacts (MI:0218) with PIP2-1 (uniprotkb:P43286) by bimolecular fluorescence complementation (MI:0809)MINT-6950816: CESA4 (uniprotkb:Q84JA6) physically interacts (MI:0218) withCESA4 (uniprotkb:Q84JA6) by membrane bound complementation assay (MI:0230)MINT-6951056, MINT-6951071, MINT-6951088, MINT-6951103: CESA7 (uniprotkb:Q9SWW6) physically interacts (MI:0218) with CESA4 (uniprotkb:Q84JA6) by bimolecular fluorescence complementation (MI:0809)MINT-6950949, MINT-6950990: CESA4 (uniprotkb:Q84JA6) physically interacts (MI:0218) with CESA8 (uniprotkb:Q8LPK5) by membrane bound complementation assay (MI:0230)MINT-6950909, MINT-6951030: CESA4 (uniprotkb:Q8LPK5) physically interacts (MI:0218) with CESA7 (uniprotkb:Q9SWW6) by membrane bound complementation assay (MI:0230)MINT-6951042: CESA4 (uniprotkb:Q84JA6) physically interacts (MI:0218) with CESA4 (uniprotkb:Q84JA6) by bimolecular fluorescence complementation (MI:0809)MINT-6951004, MINT-6951016: CESA8 (uniprotkb:Q8LPK5) physically interacts (MI:0218) with CESA7 (uniprotkb:Q9SWW6) by membrane bound complementation assay (MI:0230)MINT-6951217, MINT-6951230: CESA4 (uniprotkb:Q84JA6) physically interacts (MI:0218) with CESA8 (uniprotkb:Q8LPK5) by bimolecular fluorescence complementation (MI:0809)MINT-6951120, MINT-6951140, MINT-6951156, MINT-6951170, MINT-6951185: CESA8 (uniprotkb:Q8LPK5) physically interacts (MI:0218) withCESA7 (uniprotkb:Q9SWW6) by bimolecular fluorescence complementation (MI:0809)MINT-6951199: CESA8 (uniprotkb:Q8LPK5) physically interacts (MI:0218) withCESA8 (uniprotkb:Q8LPK5) by bimolecular fluorescence complementation (MI:0809)