Protein- and pH-dependent binding of nascent pectin and glucuronoarabinoxylan to xyloglucan in pea

 Nascent pectin and glucuronoarabinoxylan, synthesised in vitro by membrane-bound enzymes from etiolated pea (Pisum sativum L.) epicotyls, were found to bind to pea xyloglucan in a pH-dependent manner. The binding was maximum at low pH (3–4), and decreased to almost zero at pH 6. The binding was probably non-covalent and reached saturation within 5 min. Removal of the fucose residues of xyloglucan decreased the degree of binding. Removal by protease of the proteins attached to nascent pectin and glucuronoarabinoxylan greatly reduced the maximum binding and abolished the pH-dependence. The observed binding may be of considerable significance in the process of cell-wall assembly and in the control of cell extension. Received: 4 November 1999 / 22 December 1999     

Brassinosteroids, microtubules and cell elongation in Arabidopsis thaliana. II. Effects of brassinosteroids on microtubules and cell elongation in the bul1 mutant

In order to elucidate the involvement of brassinosteroids in the cell elongation process leading to normal plant morphology, indirect immunofluorescence and molecular techniques were use to study the expression of tubulin genes in the bul1-1 dwarf mutant of Arabidopsis thaliana (L.) Heynh., the characteristics of which are reported in this issue (M. Catterou et al., 2001). Microtubules were studied specifically in the regions of the mutant plant where the elongation zone is suppressed (hypocotyls and petioles), making the reduction in cell elongation evident. Indirect immunofluorescence of α-tubulin revealed that very few microtubules were present in mutant cells, resulting in the total lack of the parallel microtubule organization that is typical of elongating cells in the wild type. After brassinosteroid treatment, microtubules reorganized and became correctly oriented, suggesting the involvement of brassinosteroids in microtubule organization. Molecular analyses showed that the microtubule reorganization observed in brassinosteroid-treated bul1-1 plants did not result either from an activation of tubulin gene expression, or from an increase in tubulin content, suggesting that a brassinosteroid-responsive pathway exists which allows microtubule nucleation/organization and cell elongation without activation of tubulin gene expression. Received: 28 April 2000 / Accepted: 6 October 2000     

Fucosylation of xyloglucan: localization of the transferase in dictyosomes of pea stem cells

Microsomal membranes from elongating regions of etiolated Pisum sativum stems were separated by rate-zonal centrifugation on Renografin gradients. The transfer of labeled fucose and xylose from GDP-[¹⁴C] fucose and UDP-[¹⁴C]xylose to xyloglucan occurred mainly in dictyosomeenriched fractions. No transferase activity was detected in secretory vesicle fractions. Pulse-chase experiments using pea stem slices incubated with [³H]fucose suggest that xyloglucan chains are fucosylated and their structure completed within the dictyosomes, before being transported to the cell wall by secretory vesicles.     

Relationship between Promotion of Xyloglucan Metabolism and Induction of Elongation by Indoleacetic Acid

Auxin promotes the liberation of a xlyoglucan polymer from the cell walls of elongating pea (Pisum sativum) stem segments. The released polymer can be isolated from the polysaccharide fraction of the water-soluble portion of tissue homogenates, thus providing as assay for this kind of metabolism. Promotion of xyloglucan metabolism by auxin begins within 15 minutes of hormone presentation. The effect increases with auxin concentration in a manner similar to the hormone effect on elongation. However, the xyloglucan effect of auxin occurs perfectly normally when elongation is completely blocked by mannitol. Metabolic inhibitors and Ca²⁺, on the other hand, inhibit auxin promotion of elongation and of xyloglucan metabolism in parallel. The results suggest that the changes in xyloglucan reflect the means by which auxin modifies the cell wall to cause elongation.     

Evidence for covalent linkage between xyloglucan and acidic pectins in suspension-cultured rose cells

 Neutral xyloglucan was purified from the cell walls of suspension-cultured rose (Rosa sp. `Paul's Scarlet') cells by alkali extraction, ethanol precipitation and anion-exchange chromatography on `Q-Sepharose FastFlow'. The procedure recovered 70% of the total xyloglucan at about 95% purity in the neutral fraction. The remaining 30% of the xyloglucan was anionic, as demonstrated both by anion-exchange chromatography at pH 4.7 and by high-voltage electrophoresis at pH 6.5. Alkali did not cause neutral xyloglucan to become anionic, indicating that the anionic nature of the rose xyloglucan was not an artefact of the extraction procedure. Pre-incubation of neutral [³H]xyloglucan with any of ten non-radioactive acidic polysaccharides did not cause the radioactive material to become anionic as judged by electrophoresis, indicating that stable complexes between neutral xyloglucan and acidic polysaccharides were not readily formed in vitro. The anionic xyloglucan did not lose its charge in the presence of 8 M urea or after a second treatment with NaOH, indicating that its anionic nature was not due to hydrogen-bonding of xyloglucan to an acidic polymer. Proteinase did not affect the anionic xyloglucan, indicating that it was not associated with an acidic protein. Cellulase converted the anionic xyloglucan to the expected neutral nonasaccharide and heptasaccharide, indicating that the repeat-units of the xyloglucan did not contain acidic residues. Endo-polygalacturonase converted about 40% of the anionic xyloglucan to neutral material. Arabinanase and galactanase also converted appreciable proportions of the anionic xyloglucan to neutral material. These results show that about 30% of the xyloglucan in the cell walls of suspension-cultured rose cells exists in covalently-linked complexes with acidic pectins. Received: 5 November 1999 / Accepted: 18 January 2000     

Transport, degradation and cell wall-integration of XXFGol, a growth-regulating nonasaccharide of xyloglucan, in pea stems

When [glucitol-³H]XXFGol (a NaB³H₄-reduced xyloglucan nonasaccharide) was applied to excised shoots of pea (Pisum sativum L. cv. Progress) at the base of the epicotyl, it inhibited growth in the elongation zone, 4–5 cm distal. Experiments were conducted to discover whether such ³H-oligosaccharides are translocated intact over this distance, or whether an intercellular second messenger would have to be postulated. After 24 h, ³H from [glucitol-³H]XXFGol and [glucitol-³H]XXXGol showed U-shaped distributions, with most ³H at the base and apex of the stem. Radioactivity from [fucosyl-³H]XXFG and [xylosyl-³H]XXFG also moved acropetally, but did not concentrate at the apex, possibly owing to removal from the transpiration stream of fucose and xylose formed by partial hydrolysis of XXFG en route. When 10⁻⁷ M [glucitol-³H]XXFGol was supplied, about 14 fmol ·  seedling^–1 of apparently intact [³H]XXFGol was extractable from the elongation zone after 24 h. Larger amounts of degradation products were extractable (including free [³H]glucitol) and some wall-bound ³H-hemicellulose was present (presumably formed by the oligosaccharides acting as acceptor substrates for transglycosylation). We conclude that biologically active oligosaccharides of xyloglucan can move through the stem acropetally and that they are maintained at low steady-state concentrations by both hydrolysis and transglycosylation. Received: 1 April 1997 / Accepted: 28 May 1997     

Pea xyloglucan and cellulose : I. Macromolecular organization

A macromolecular complex composed of xyloglucan and cellulose was obtained from elongating regions of etiolated pea (Pisum sativum L. var. Alaska) stems. Xyloglucan could be solubilized by extraction of this complex with 24% KOH-0.1% NaBH₄ or by extended treatment with endo-1,4-β-glucanase. The polysaccharide was homogeneous by ultracentrifugal analysis and gel filtration on Sepharose CL-6B, molecular weight 330,000. The structure of pea xyloglucan was examined by fragmentation analysis of enzymic hydrolysates, methylation analysis, and precipitation tests with fucose- or galactose-binding lectins. The polysaccharide was composed of equal amounts of two subunits, a nonasaccharide (glucose/xylose/galactose/fucose, 4:3:1:1) and a heptasaccharide (glucose/xylose, 4:3), which appeared to be distributed at random, but primarily in alternating sequence. The xyloglucan:cellulose complex was examined by light microscopy using iodine staining, by radioautography after labeling with [³H]fucose, by fluorescence microscopy using a fluorescein-lectin (fucose-binding) as probe, and by electron microscopy after shadowing. The techniques all demonstrated that the macromolecule was present in files of cell shapes, referred to here as cell-wall `ghosts,' in which xyloglucan was localized both on and between the cellulose microfibrils. Since the average chain length of pea xyloglucan was many times the diameter of cellulose microfibrils, it could introduce cross-links by binding to adjacent fibrils and thereby contribute rigidity to the wall.     

Localization of Xyloglucan in the Macromolecular Complex Composed of Xyloglucan and Cellulose in Pea Stems

A macromolecular complex composed of xyloglucan and cellulosewas isolated from elongating regions of stems of etiolated pea(Pisum sativum L. var Alaska) seedlings and binding of a xyloglucan-specificantibody was examined after treatment of the complex with endo-1,4-ß-glucanaseor 24% KOH. The antibody bound to the complex but the extentof binding was reduced after treatment of the complex with endo-1,4-ß-glucanaseand was hardly detectable after treatment with 24% KOH. Themolecular weight of the xyloglucan that remained (5%) in theß-glucanase-treated complexes was less than 9,200.Pea xyloglucan was allowed to bind to enzymeand alkali-treatedcomplexes to generaly reconstituted complexes. The amount ofthe antibody that bound to each type of reconstituted complexwas similar but was much lower than that bound to the nativecomplex. Immunogold labeling indicated that most of the antigenwas widely distributed between microfibrils in the native complex,whereas the antigen appeared to be confined to the microfibrilsin the reconstituted complexes. These findings suggest thata part of each xyloglucan molecule is strongly associated withcellulose microfibrils while the rest is free of the microfibrilsin the native complex. ¹This work was supported in part by a grant from the YamadaScience Foundation.     

α-Expansins in the semiaquatic ferns Marsilea quadrifolia and Regnellidium diphyllum: evolutionary aspects and physiological role in rachis elongation

To investigate the evolutionary history of expansins and their role in cell elongation in early land plants, we isolated two α-expansin genes, Mq-EXP1 and Rd-EXP1, respectively, from the semiaquatic ferns Marsilea quadrifolia L. and Regnellidium diphyllum Lindm. The deduced amino acid sequences of the fern expansins exhibit a high degree of identity to those of seed plants, showing that expansin genes were conserved during the evolution of vascular plants. Gel-blot analysis of M. quadrifolia and R. diphyllum genomic DNA indicated that, in both ferns, α-expansins are encoded by multigene families. Expression of α-expansin genes probed with Mq-EXP1 was confined to the elongating region of the Marsilea rachis. Cell-wall proteins of M.␣quadrifolia induced in-vitro extension of acidified cucumber cell walls. In R. diphyllum, expression of Rd-EXP1 increased when elongation of the rachis was enhanced by submergence or ethylene. These results indicate that α-expansins act as wall-loosening proteins in ferns, as has been proposed for angiosperms. In addition, Rd-EXP1 may play a role in mediating elongation of the rachis in submerged plants. Received: 7 March 2000 / Accepted: 29 April 2000     

Roles of the fructans from leaf sheaths and from the elongating leaf bases in the regrowth following defoliation of Lolium perenne L

The study of carbohydrate metabolism in perennial ryegrass (Lolium perenne L. cv. Bravo) during the first 48 h of regrowth showed that fructans from elongating leaf bases were hydrolysed first whereas fructans in mature leaf sheaths were degraded only after a lag of 1.5 h. In elongating leaf bases, the decline in fructan content occurred not only in the differentiation zone (30–60 mm from the leaf base), but also in the growth zone. Unlike other soluble carbohydrates, the net deposition rate of fructose remained positive and even rose during the first day following defoliation. The activity of fructan exohydrolase (FEH; EC 3.2.1.80) was maximal in the differentiation zone before defoliation and increased in all segments, but peaked in the growth zone after defoliation. These data strongly indicate that fructans stored in the leaf growth zone were hydrolysed and recycled in that zone to sustain the refoliation immediately after defoliation. Despite the depletion of carbohydrates, leaves of defoliated plants elongated at a significantly higher rate than those of undefoliated plants, during the first 10 h of regrowth. This can be partly attributed to the transient increase in water and nitrate deposition rate. The results are discussed in relation to defoliation tolerance. Received: 16 June 2000 / Accepted: 17 October 2000     

Soybean leghemoglobin targeted to potato chloroplasts influences growth and development of transgenic plants

 Potato tubers were transformed with a chimeric gene made by the fusion of the soybean leghemoglobin encoding gene (lba) with the chloroplastic targeting sequence from Rubisco. This construct was placed under the control of the strong constitutive 35S promoter and the 3′ nontranslated region of Rubisco from pea. Leghemoglobin expression on kanamycin-resistant plants was monitored by RT-PCR. Furthermore, immunodetection of subcellular fractions of transgenic plants revealed that leghemoglobin was imported and correctively processed inside the organelle. In addition, analysis of transgenic plants revealed reduced growth and decreased tuber production compared with the untransformed plants. It is suggested that leghemoglobin expression in potato chloroplasts interferes with aerobic metabolism, leading to physiological and morphological changes. Received: 20 December 1999 / Revision received: 28 May 2000 / Accepted: 16 June 2000     

The relationship between xyloglucan endotransglycosylase and in-vitro cell wall extension in cucumber hypocotyls

It has been proposed that cell wall loosening during plant cell growth may be mediated by the endotransglycosylation of load-bearing polymers, specifically of xyloglucans, within the cell wall. A xyloglucan endotransglycosylase (XET) with such activity has recently been identified in several plant species. Two cell wall proteins capable of inducing the extension of plant cell walls have also recently been identified in cucumber hypocotyls. In this report we examine three questions: (1) Does XET induce the extension of isolated cell walls? (2) Do the extension-inducing proteins possess XET activity? (3) Is the activity of the extension-inducing proteins modulated by a xyloglucan nonasaccharide (Glc₄-Xyl₃-Gal₂)? We found that the soluble proteins from growing cucumber (cucumis sativum L.) hypocotyls contained high XET activity but did not induce wall extension. Highly purified wall-protein fractions from the same tissue had high extension-inducing activity but little or no XET activity. The XET activity was higher at pH 5.5 than at pH 4.5, while extension activity showed the opposite sensitivity to pH. Reconstituted wall extension was unaffected by the presence of a xyloglucan nonasaccharide (Glc₄-Xyl₃-Gal₂), an oligosaccharide previously shown to accelerate growth in pea stems and hypothesized to facilitate growth through an effect on XET-induced cell wall loosening. We conclude that XET activity alone is neither sufficient nor necessary for extension of isolated walls from cucumber hypocotyls.     

Xyloglucan−pectin linkages are formed intra-protoplasmically,contribute to wall-assembly,and remain stable in the cell wall

We tested two hypotheses for the mechanism by which xyloglucan–pectin covalent bonds are formed in Arabidopsis cell cultures. Hypothesis 1 proposed hetero-transglycosylation, with xyloglucan as donor substrate and a rhamnogalacturonan-I (RG-I) side-chain as acceptor. We looked for enzyme activities that catalyse this reaction using α-(1→5)-l-[³H]arabino- or β-(1→4)-d-[³H]galacto-oligosaccharides as model acceptor substrates. The ³H-oligosaccharides were supplied (with or without added xyloglucans) to living Arabidopsis cell-cultures, permeabilised cells, cell-free extracts, or four authentic XTHs. No hetero-transglycosylation occurred. Therefore, we cannot support hypothesis 1. Hypothesis 2 proposed that some xyloglucan is manufactured de novo as a side-chain on RG-I. To test this, we pulse-labelled Arabidopsis cell-cultures with [³H]arabinose and monitored the radiolabelling of anionic (pectin-bonded) xyloglucan, which was resolved from free xyloglucan by ion-exchange chromatography. [³H]Xyloglucan–pectin complexes were detectable <4 min after [³H]arabinose feeding, which is shorter than the transit-time for polysaccharide secretion, indicating that xyloglucan–pectin bonds were formed intra-protoplasmically. Thereafter, the proportion of the wall-bound [³H]xyloglucan that was anionic remained almost constant at ∼50% for ≥6 days, showing that the xyloglucan–pectin bond was stable in vivo. Some [³H]xyloglucan was rapidly sloughed into the medium instead of becoming wall-bound. Only ∼30% of the sloughed [³H]xyloglucan was anionic, indicating that bonding to pectin promoted the integration of xyloglucan into the wall. We conclude that ∼50% of xyloglucan in cultured Arabidopsis cells is synthesised on a pectic primer, then secreted into the apoplast, where the xyloglucan–pectin bonds are stable and the pectic moiety aids wall-assembly.     

Fructose 2,6-bisphosphate activates pyrophosphate: fructose-6-phosphate 1-phosphotransferase and increases triose phosphate to hexose phosphate cycling in heterotrophic cells

The aim of this work was to establish the influence of fructose 2,6-bisphosphate (Fru-2,6-P₂) on non-photosynthetic carbohydrate metabolism in plants. Heterotrophic callus lines exhibiting elevated levels of Fru-2,6-P₂ were generated from transgenic tobacco (Nicotiana tabacum L.) plants expressing a modified rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Lines containing increased amounts of Fru-2,6-P₂ had lower levels of hexose phosphates and higher levels of 3-phosphoglycerate than the untransformed control cultures. There was also a greater redistribution of label into the C6 position of sucrose and fructose, following incubation with [1-¹³C]glucose, in the lines possessing the highest amounts of Fru-2,6-P₂, indicating a greater re-synthesis of hexose phosphates from triose phosphates in these lines. Despite these changes, there were no marked differences between lines in the metabolism of ¹⁴C-substrates, the rate of oxygen uptake, carbohydrate accumulation or nucleotide pool sizes. These data provide direct evidence that physiologically relevant changes in the level of Fru-2,6-P₂ can affect pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) activity in vivo, and are consistent with PFP operating in a net glycolytic direction in the heterotrophic culture. However, the results also show that activating PFP has little direct effect on heterotrophic carbohydrate metabolism beyond increasing the rate of cycling between hexose phosphates and triose phosphates. Received: 29 March 2000 / Accepted: 13 June 2000     

A Xyloglucan-Specific Endo-1,4-[beta]-Glucanase Isolated from Auxin-Treated Pea Stems

A xyloglucan-specific endo-1,4-[beta]-glucanase was isolated from the apoplast fraction of auxin-treated pea (Pisum sativum) stems, in which both the rate of stem elongation and the amount of xyloglucan solubilized were high. The enzyme was purified to apparent homogeneity by sequential cation-exchange chromatographies, affinity chromatography, and gel filtration. The purified enzyme gave a single protein band on sodium dodecyi sulfate-polyacrylamide gel electrophoresis, and the molecular size was determined to be 77 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 70 kD by gel filtration. The isoelectric point was about 8.1. The enzyme specifically cleaved the 1,4-[beta]-glucosyl linkages of the xyloglucan backbone to yield mainly nona- and heptasaccharides but did not hydrolyze carboxymethylcellulose, swollen cellulose, and (1->3, 1->4)-[beta]-glucan. By hydrolysis, the average molecular size of xyloglucan was decreased from 50 to 20 kD with new reducing chain ends in the lower molecular size fractions. This suggests that the enzyme has endo-1,4-[beta]-glucanase activity against xyloglucan. In conclusion, a xyloglucan-specific endo-1,4-[beta]-glucanase with an activity that differs from the activities of cellulase and xyloglucan endotransglycosylase has been isolated from elongating pea stems.     

Functional characterisation of Nicotiana tabacum xyloglucan endotransglycosylase (NtXET-1): generation of transgenic tobacco plants and changes in cell wall xyloglucan

To study the function of xyloglucan endotransglycosylase (XET) in vivo we isolated, a tomato (Lycopersicon esculentum Mill.) XET cDNA (GenBank AA824986) from the homologous tobacco (Nicotiana tabacum L.) clone named NtXET-1 (Accession no. D86730). The expression pattern revealed highest levels of NtXET-1 mRNA in organs highly enriched in vascular tissue. The levels of NtXET-1 mRNA decreased in midribs with increasing age of leaves. Increasing leaf age was correlated with an increase in the average molecular weight (MW) of xyloglucan (XG) and a decrease in the relative growth rates of leaves. Transgenic tobacco plants with reduced levels of XET activity were created to further study the biochemical consequences of reduced levels of NtXET-1 expression. In two independent lines, total XET activity could be reduced by 56% and 37%, respectively, in midribs of tobacco plants transformed with an antisense construct. The decreased activity led to an increase in the average MW of XG by at least 20%. These two lines of evidence argue for NtXET-1 being involved in the incorporation of small XG molecules into the cell wall by transglycosylation. Reducing the incorporation of small XG molecules will result in a shift towards a higher average MW. The observed reduction in NtXET-1 expression and increase in the MW of XG in older leaves might be associated with strengthening of cell walls by reduced turnover and hydrolysis of XG. Received: 24 January 2000 / Accepted: 21 July 2000     

Acid-induced elongation of Reynoutria stems requires tissue stresses

Measurements of the elongation rate of strips of outer tissue composed of epidermis and collenchyma, peeled from the elongating internodes of Reynoutria japonica Houtt. (Japanese knotweed) and stretched in buffer, showed that the rate depended on pH and the stretching force. At pH 6.8, elongation was barely perceptible in the force range studied, but the rate of elongation increased rapidly with the force at pH 5.0 (acid-induced elongation). The yield threshold stress at pH 5.0 amounted to 1.8×10⁶ N m^?2. It was three times higher than the osmotic pressure in the outer tissue, but was lower than the average tensile tissue stress in this tissue in intact stems. We infer that tensile tissue stress in the outer tissue is required for the manifestation of acid-induced elongation of this tissue in situ.     

A Homologue of EGL1 Encoding Endo-1,4-β-glucanase in Elongating Pea Stems

A gene (EGL2) encoding an endo-1,4-β-glucanase in peas has been cloned as a homologue of EGL1. EGL2 encodes a polypeptide of 506 amino acids, including a 24-mer putative signal polypeptide. The gene product contains a domain conserved in endo-1,4-β-glucanase (family 9) showing 60% amino acid identity to EGL1. EGL2 mRNA was accumulated only in the elongating regions of pea stems, although EGL1 mRNA was abundant in both elongating and non-elongating tissues. However, the level of EGL2 mRNA was not increased by the treatment with sucrose and auxin in pea segments. These results suggest that the expression of EGL2 either requires the presence of other factors related to the auxin effect or occurs independent of auxin in the elongating pea stems.     

The Involvement of Cytokinin Oxidase/Dehydrogenase and Zeatin Reductase in Regulation of Cytokinin Levels in Pea (Pisum sativum L.) Leaves

Cytokinin metabolism in plants is very complex. More than 20 cytokinins bearing isoprenoid and aromatic side chains were identified by high performance liquid chromatography-mass spectrometry (HPLC-MS) in pea (Pisum sativum L. cv. Gotik) leaves, indicating diverse metabolic conversions of primary products of cytokinin biosynthesis. To determine the potential involvement of two enzymes metabolizing cytokinins, cytokinin oxidase/dehydrogenase (CKX, EC 1.5.99.12) and zeatin reductase (ZRED, EC 1.3.1.69), in the control of endogenous cytokinin levels, their in vitro activities were investigated in relation to the uptake and metabolism of [2−³H]trans-zeatin ([2−³H]Z) in shoot explants of pea. Trans-zeatin 9-riboside, trans-zeatin 9-riboside-5′-monophosphate and cytokinin degradation products adenine and adenosine were detected as predominant [2−³H]Z metabolites during 2, 5, 8, and 24 h incubation. Increasing formation of adenine and adenosine indicated extensive degradation of [2−³H]Z by CKX. High CKX activity was confirmed in protein preparations from pea leaves, stems, and roots by in vitro assays. Inhibition of CKX by dithiothreitol (15 mM) in the enzyme assays revealed relatively high activity of ZRED catalyzing conversion of Z to dihydrozeatin (DHZ) and evidently competing for the same substrate cytokinin (Z) in protein preparations from pea leaves, but not from pea roots and stems. The conversion of Z to DHZ by pea leaf enzyme was NADPH dependent and was significantly inhibited or completely suppressed in vitro by diethyldithiocarbamic acid (DIECA; 10 mM). Relations of CKX and ZRED in the control of cytokinin levels in pea leaves with respect to their potential role in establishment and maintenance of cytokinin homeostasis in plants are discussed.