Auxin-regulated genes encoding cell wall-modifying proteins are expressed during early tomato fruit growth

An expansin gene, LeExp2, was isolated from auxin-treated, etiolated tomato (Lycopersicon esculentum cv T5) hypocotyls. LeExp2 mRNA expression was restricted to the growing regions of the tomato hypocotyl and was up-regulated during incubation of hypocotyl segments with auxin. The pattern of expression of LeExp2 was also studied during tomato fruit growth, a developmental process involving rapid cell enlargement. The expression of genes encoding a xyloglucan endotransglycosylase (LeEXT1) and an endo-1, 4-beta-glucanase (Cel7), which, like LeExp2, are auxin-regulated in etiolated hypocotyls (C. Catalá, J.K.C. Rose, A.B. Bennett [1997] Plant J 12: 417-426), was also studied to examine the potential for synergistic action with expansins. LeExp2 and LeEXT1 genes were coordinately regulated, with their mRNA accumulation peaking during the stages of highest growth, while Cel7 mRNA abundance increased and remained constant during later stages of fruit growth. The expression of LeExp2, LeEXT1, and Cel7 was undetectable or negligible at the onset of and during fruit ripening, which is consistent with a specific role of these genes in regulating cell wall loosening during fruit growth, not in ripening-associated cell wall disassembly.     

Characterization of a Tomato Xyloglucan Endotransglycosylase Gene That Is Down-Regulated by Auxin in Etiolated Hypocotyls

The reorganization of the cellulose-xyloglucan matrix is proposed to serve as an important mechanism in the control of strength and extensibility of the plant primary cell wall. One of the key enzymes associated with xyloglucan metabolism is xyloglucan endotransglycosylase (XET), which catalyzes the endocleavage and religation of xyloglucan molecules. As with other plant species, XETs are encoded by a gene family in tomato (Lycopersicon esculentum cv T5). In a previous study, we demonstrated that the tomato XET gene LeEXT was abundantly expressed in the rapidly expanding region of the etiolated hypocotyl and was induced to higher levels by auxin. Here, we report the identification of a new tomato XET gene, LeXET2, that shows a different spatial expression and diametrically opposite pattern of auxin regulation from LeEXT. LeXET2 was expressed more abundantly in the mature nonelongating regions of the hypocotyl, and its mRNA abundance decreased dramatically following auxin treatment of etiolated hypocotyl segments. Analysis of the effect of several plant hormones on LeXET2 expression revealed that the inhibition of LeXET2 mRNA accumulation also occurred with cytokinin treatment. LeXET2 mRNA levels increased significantly in hypocotyl segments treated with gibberellin, but this increase could be prevented by adding auxin or cytokinin to the incubation media. Recombinant LeXET2 protein obtained by heterologous expression in Pichia pastoris exhibited greater XET activity against xyloglucan from tomato than that from three other species. The opposite patterns of expression and differential auxin regulation of LeXET2 and LeEXT suggest that they encode XETs with distinct roles during plant growth and development.     

A plasma membrane-bound putative endo-1,4-beta-D-glucanase is required for normal wall assembly and cell elongation in Arabidopsis.

Endo-1,4-beta-D-glucanases (EGases) form a large family of hydrolytic enzymes in prokaryotes and eukaryotes. In higher plants, potential substrates in vivo are xyloglucan and non-crystalline cellulose in the cell wall. Gene expression patterns suggest a role for EGases in various developmental processes such as leaf abscission, fruit ripening and cell expansion. Using Arabidopsis thaliana genetics, we demonstrate the requirement of a specialized member of the EGase family for the correct assembly of the walls of elongating cells. KORRIGAN (KOR) is identified by an extreme dwarf mutant with pronounced architectural alterations in the primary cell wall. The KOR gene was isolated and encodes a membrane-anchored member of the EGase family, which is highly conserved between mono- and dicotyledonous plants. KOR is located primarily in the plasma membrane and presumably acts at the plasma membrane-cell wall interface. KOR mRNA was found in all organs examined, and in the developing dark-grown hypocotyl, mRNA levels were correlated with rapid cell elongation. Among plant growth factors involved in the control of hypocotyl elongation (auxin, gibberellins and ethylene) none significantly influenced KOR-mRNA levels. However, reduced KOR-mRNA levels were observed in det2, a mutant deficient for brassinosteroids. Although the in vivo substrate remains to be determined, the mutant phenotype is consistent with a central role for KOR in the assembly of the cellulose-hemicellulose network in the expanding cell wall.     

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.     

Auxin-regulated gene expression

During the 1960s a wide range of studies provided an information base that led to the suggestion that auxin-regulated cell processes--especially cell elongation--may be mediated by auxin-regulated gene expression. Indirect evidence from our work, based on the influence of inhibitors of RNA synthesis (e.g. actinomycin D) and of protein synthesis (e.g. cycloheximide) on auxin-induced cell elongation, coupled with correlations of the influence of auxin on RNA synthesis and cell elongation, provided the basis for this suggestion. With the availability of techniques for DNA-DNA and DNA-RNA hybridization, mRNA isolation-translation, in vitro 2D gel analysis of the translation products, and ultimately the cloning by recombinant DNA technologies of genomic DNA and copy DNAs (cDNAs) made to poly(A)+ mRNAs, we and others have provided direct evidence for the influence of auxin on the expression of a few genes (i.e. poly(A)+ RNA levels). Our laboratory has provided evidence for auxin's both down-regulating and up-regulating the level of a few poly(A)+ mRNAs out of a population of about 4 X 10(4) sequences that are not significantly affected by auxin. In our studies on auxin-regulated cell elongation, two cDNA clones (pJCW1 and pJCW2) were isolated which corresponded to poly(A)+ mRNAs that responded during growth transitions in a way consistent with a potential role of their protein products in cell elongation. These mRNAs are most abundant in the elongating zone of the soybean hypocotyl. Upon excision and incubation in the absence of auxin, these mRNAs deplete in concert with a decreasing rate of cell elongation. Addition of auxin to the medium results in both increased levels of these mRNAs and enhanced rates of cell elongation. These mRNAs do not deplete if auxin is added to the medium at the onset of excised incubation, and cell elongation rates remain high. We have isolated and sequenced genomic clones that are homologous to these cDNAs. Of the two genes sequenced, both genes are members of small multigene families. There are regions of high amino acid homology even though the nucleotide sequences are sufficiently different in these regions for cross-hybridization of the clones not to be observed. More recently others, especially Guilfoyle's laboratory, have shown that auxin selectively and rapidly influences the level of certain mRNAs and proteins. We have worked on other gene systems such as ribosomal proteins and possible cell wall proteins that are responsive to auxin; again the nature of regulation of expression of these genes is not known.(ABSTRACT TRUNCATED AT 400 WORDS)     

A revised architecture of primary cell walls based on biomechanical changes induced by substrate-specific endoglucanases

Xyloglucan is widely believed to function as a tether between cellulose microfibrils in the primary cell wall, limiting cell enlargement by restricting the ability of microfibrils to separate laterally. To test the biomechanical predictions of this "tethered network" model, we assessed the ability of cucumber (Cucumis sativus) hypocotyl walls to undergo creep (long-term, irreversible extension) in response to three family-12 endo-β-1,4-glucanases that can specifically hydrolyze xyloglucan, cellulose, or both. Xyloglucan-specific endoglucanase (XEG from Aspergillus aculeatus) failed to induce cell wall creep, whereas an endoglucanase that hydrolyzes both xyloglucan and cellulose (Cel12A from Hypocrea jecorina) induced a high creep rate. A cellulose-specific endoglucanase (CEG from Aspergillus niger) did not cause cell wall creep, either by itself or in combination with XEG. Tests with additional enzymes, including a family-5 endoglucanase, confirmed the conclusion that to cause creep, endoglucanases must cut both xyloglucan and cellulose. Similar results were obtained with measurements of elastic and plastic compliance. Both XEG and Cel12A hydrolyzed xyloglucan in intact walls, but Cel12A could hydrolyze a minor xyloglucan compartment recalcitrant to XEG digestion. Xyloglucan involvement in these enzyme responses was confirmed by experiments with Arabidopsis (Arabidopsis thaliana) hypocotyls, where Cel12A induced creep in wild-type but not in xyloglucan-deficient (xxt1/xxt2) walls. Our results are incompatible with the common depiction of xyloglucan as a load-bearing tether spanning the 20- to 40-nm spacing between cellulose microfibrils, but they do implicate a minor xyloglucan component in wall mechanics. The structurally important xyloglucan may be located in limited regions of tight contact between microfibrils.     

Differential expression of AtXTH17, AtXTH18, AtXTH19 and AtXTH20 genes in Arabidopsis roots. Physiological roles in specification in cell wall construction

Xyloglucan endotransglucosylase/hydrolases (XTHs) are a class of enzymes that are capable of splitting and reconnecting xyloglucan molecules, and are implicated in the construction and restructuring of the cellulose/xyloglucan framework. Thirty-three members of the XTH gene family are found in the genome of Arabidopsis thaliana, but their roles remain unclear. Here, we describe the tissue-specific and growth stage-dependent expression profiles of promoter::GUS fusion constructs for four Arabidopsis XTH genes, AtXTH17, AtXTH18, AtXTH19 and AtXTH20, which are phylogenetically closely related to one another. AtXTH17 and AtXTH18 were expressed in all cell types in the elongating and differentiating region of the root, while AtXTH19 was expressed in the apical dividing and elongating regions, as well as in the differentiation zone, and was up-regulated by auxin. In contrast, AtXTH20 was expressed specifically in vascular tissues in the basal mature region of the root. This expression analysis also disclosed cis-regulatory sequences that are conserved among the four genes, and are responsible for the root-specific expression profile. These results indicate that the four XTH genes, which were generated by gene duplication, have diversified their expression profile within the root in such a way as to take responsibility for particular physiological roles in the cell wall dynamics.     

The cell wall-modifying xyloglucan endotransglycosylase/hydrolase LeXTH1 is expressed during the defence reaction of tomato against the plant parasite Cuscuta reflexa

A suppressive subtractive hybridization technique was used to identify genes, which were induced during the early phases of the interaction between dodder (Cuscuta reflexa), a phanerogamic parasite, and its incompatible host plant tomato. One of the identified genes encodes a tomato xyloglucan endotransglycosylase/hydrolase (XTH)--an enzyme involved in cell wall elongation and restructuring. The corresponding LeXTH1 mRNA accumulated 6 h after attachment of the parasite. In contrast, wounding did not influence the expression level. Subsequent to LeXTH1 mRNA accumulation, an increase in XTH activity at the infection sites as well as in adjacent tissues was observed. The effect of IAA on LeXTH1 expression was analyzed because the concentration of this phytohormone is known to increase in the tomato tissue during the interaction with the parasite. LeXTH1 mRNA accumulation was in fact induced by external application of auxin. However, in the auxin-insensitive tomato mutant diageotropica, Cuscuta induced LeXTH1-mRNA accumulated with a time course similar to wild type tomato. Thus, auxin appears not to be an essential signal for infection-induced LeXTH1 activation. Our data suggest a role for xyloglucan transglycosylation in defence reactions associated with the incompatible tomato- Cuscuta interaction.     

Growth capacity and molecular mass distribution of cell wall polysaccharides along the hypocotyl of Pinus pinaster

The changes in the endogenous growth as well as in the cell wall composition were studied along the hypocotyl of Pinus pinaster Aiton. Cell elongation decreased as the distance from the cotyledonary node increased. Pectic polysaccharides underwent an important depolymerization accompanied by a decrease in their uronic acid content from the apical to basal region of the hypocotyl. Additionally, the molecular mass of pectic polysaccharides strongly decreased from the apical to the basal regions. Watersoluble hemicellulosic polysaccharides extracted with 4% KOH decreased notably from the cotyledonary node towards the base, while water-soluble polysaccharides extracted with 24% KOH showed few differences along the hypocotyl. The molecular mass of xyloglucan present in both hemicellulosic fractions was lower in the upper hypocotyl region as compared with the basal region. These findings are in agreement with an active xyloglucan depolymerization in the upper region as would be expected in a region exhibiting very active growth.     

Auxin-regulated Wall Loosening and Sustained Growth in Elongation

It is proposed that auxin regulates and coordinates both wall loosening and the supply of wall materials in elongation. The tenets of the proposal allowed testable predictions. It was determined that, if the cell walls of Glycine max L. var. Wayne hypocotyl segments are maintained in a loosened state (by excising the segments directly into pH 4 medium), exogenous auxin induced only the second response. It was also predicted and confirmed that elongating systems, e.g. pea epicotyl, with certain early auxin-induced growth kinetics (an initial high non-steady-state rate followed immediately by a drop to a lower steady-state rate) would show a transient second response (in addition to the usual transient first response) when stimulated by pH 4 medium. Finally, it is pointed out that recent results which establish the existence of auxin-induced elongation-associated proteins support the proposition that auxin coordinates wall loosening and the supply of wall materials in elongation.     

Growth and stomata development of Arabidopsis hypocotyls are controlled by gibberellins and modulated by ethylene and auxins

The plant hormones gibberellin (GA), ethylene and auxin can promote hypocotyl elongation of Arabidopsis seedlings grown in the light on a low nutrient medium (LNM). In this study, we used hypocotyl elongation as a system to investigate interactions between GA and ethylene or auxin and analysed their influence on the development of stomata in the hypocotyl. When applied together, GA and ethylene or auxin exerted a synergistic effect on hypocotyl elongation. Stimulated cell elongation is the main cause of hypocotyl elongation. Furthermore, hypocotyls treated with GA plus either ethylene or auxin show an increased endoreduplication. In addition, a small but significant increase in cell number was observed in the cortical cell files of hypocotyls treated with ethylene and GA together. However, studies with transgenic seedlings expressing CycB1::uidA genes revealed that cell division in the hypocotyl occurs only in the epidermis and mainly to form stomata, a process strictly regulated by hormones. Stomata formation in the hypocotyl is induced by the treatment with either GA or ethylene. The effect of GA could be strongly enhanced by the simultaneous addition of ethylene or auxin to the growth medium. Gibberellin is the main signal inducing stomata formation in the hypocotyl. In addition, this signal regulates hypocotyl elongation and is modulated by ethylene and auxin. The implication of these three hormones in relation to cell division and stomata formation is discussed.     

Cell wall development in maize coleoptiles

The physical bases for enhancement of growth rates induced by auxin involve changes in cell wall structure. Changes in the chemical composition of the primary walls during maize (Zea mays L. cv WF9 × Bear 38) coleoptile development were examined to provide a framework to study the nature of auxin action. This report documents that the primary walls of maize cells vary markedly depending on developmental state; polymers synthesized and deposited in the primary wall during cell division are substantially different from those formed during cell elongation.

The embryonal coleoptile wall is comprised of mostly glucuronoarabinoxylan (GAX), xyloglucan, and polymers enriched in 5-arabinosyl linkages. During development, both GAX and xyloglucan are synthesized, but the 5-arabinosyls are not. Rapid coleoptile elongation is accompanied by synthesis of a mixed-linked glucan that is nearly absent from the embryonal wall. A GAX highly substituted with mostly terminal arabinofuranosyl units is also synthesized during elongation and, based on pulse-chase studies, exhibits turnover possibly to xylans with less substitution via loss of the arabinosyl and glucuronosyl linkages.

     

Why hypocotyl extension mutants need to be characterized at the cell level: a case study of axr3-1

Since the discovery of auxin, a debate has taken place as to whether the auxin distribution in elongating organs can account for the distinctive cell elongation profiles that have been found. In an attempt to address this important issue, the elongation profiles of cells have been compared in the hypocotyls of wild-type and auxin-hypersensitive axr3-1 Arabidopsis Columbia ecotype seedlings. Clear differences in cell elongation profiles were found in the two types of seedling, whether they were light- or dark-grown. However, it was not possible unambiguously to ascribe the cell elongation profile differences to the proposition that the axr3-1 mutation causes the hypocotyl to be hypersensitive to auxin. The possibility that the abnormal hypocotyl elongation profile of the mutant was a secondary effect, consequent on a more fundamental effect of the axr3-1 mutation, is considered. It is clear from this study that cell elongation and its control needs to be studied at the cell, and not the organ, level. To characterize a mutant as having a short, or long, hypocotyl is inadequate. To determine which factors control the timing and the magnitude of cell elongation requires the demonstration of correlations between the growth rate of cells and their content of regulating substances or their sensitivity to that substance. Studies of the cell elongation profiles of the many hypocotyl length mutants could also be a very effective means of probing the co-ordination of root and shoot elongation.     

Characterization of cross-links between cellulose microfibrils, and their occurrence during elongation growth in pea epicotyl

The occurrence and chemical nature of the cross-links between cellulose microfibrils in outer epidermal cell walls in Pisum sativum cv. Alaska was investigated by rapid-freezing and deep-etching techniques coupled with chemical and enzymatic treatments. The cell wall in the elongating region of epidermal cells was characterized by the absence of the cross-links, while in the elongated region, the cell wall was characterized by the presence of cross-links. The cross-links remained in the cell wall of the elongated region after treatment with SDS electrophoresis sample buffer and treatment with 4% potassium hydroxide. After treatment with endo-1,4-beta-glucanase, which fragments xyloglucan, the cross-links were remarkably reduced from the cell wall of the elongated region. The endoglucanase treatment also reduced immunogold labeling of xyloglucan in the cell wall. The endoglucanase hydrolysate from the cell wall fraction of the elongated region gave spots of oligosaccharides in thin layer chromatography, which were identical to the spots of xyloglucan oligosaccharides produced by xyloglucanase from both the cell wall fraction and tamarind xyloglucan. These results indicate that the cross-links are made of xyloglucan. We discussed the possibility of cross-links involved in the control of mechanical properties of the cell wall.     

Promotion of Xyloglucan Metabolism by Acid pH

Like indoleacetic acid, buffers of acidic pH, which stimulate elongation of pea (Pisum sativum var. Alaska) stem tissue, induce the appearance within the tissue of a watersoluble xyloglucan polymer that probably arises from previously deposited wall material. Neutral pH buffers, which inhibit the elongation response to indoleacetic acid in this tissue, inhibit indoleacetic acid-induced increase in soluble xyloglucan. The findings provide further evidence that release of soluble xyloglucan from the cell walls of pea results from the biochemical action on the cell wall that is responsible for wall extension. The data also indicate that treatment of tissue with either auxin or acidic pH has a similar biochemical effect on the cell wall. This is consistent with the H⁺ secretion theory of auxin action.