Stephen C. Fry

Arabidopsis TCH4, regulated by hormones and the environment, encodes a xyloglucan endotransglycosylase.

Adaptation of plants to environmental conditions requires that sensing of external stimuli be linked to mechanisms of morphogenesis. The Arabidopsis TCH (for touch) genes are rapidly upregulated in expression in response to environmental stimuli, but a connection between this molecular response and developmental alterations has not been established. We identified TCH4 as a xyloglucan endotransglycosylase by sequence similarity and enzyme activity. Xyloglucan endotransglycosylases most likely modify cell walls, a fundamental determinant of plant form. We determined that TCH4 expression is regulated by auxin and brassinosteroids, by environmental stimuli, and during development, by a 1-kb region. Expression was restricted to expanding tissues and organs that undergo cell wall modification. Regulation of genes encoding cell wall-modifying enzymes, such as TCH4, may underlie plant morphogenetic responses to the environment.

In Vivo Cell Wall Loosening by Hydroxyl Radicals during Cress Seed Germination and Elongation Growth

Loosening of cell walls is an important developmental process in key stages of the plant life cycle, including seed germination, elongation growth, and fruit ripening. Here, we report direct in vivo evidence for hydroxyl radical (·OH)-mediated cell wall loosening during plant seed germination and seedling growth. We used electron paramagnetic resonance spectroscopy to show that ·OH is generated in the cell wall during radicle elongation and weakening of the endosperm of cress (Lepidium sativum; Brassicaceae) seeds. Endosperm weakening precedes radicle emergence, as demonstrated by direct biomechanical measurements. By 3H fingerprinting, we showed that wall polysaccharides are oxidized in vivo by the developmentally regulated action of apoplastic ·OH in radicles and endosperm caps: the production and action of ·OH increased during endosperm weakening and radicle elongation and were inhibited by the germination-inhibiting hormone abscisic acid. Both effects were reversed by gibberellin. Distinct and tissue-specific target sites of ·OH attack on polysaccharides were evident. In vivo ·OH attack on cell wall polysaccharides were evident not only in germinating seeds but also in elongating maize (Zea mays; Poaceae) seedling coleoptiles. We conclude that plant cell wall loosening by ·OH is a controlled action of this type of reactive oxygen species.

In Vivo Colocalization of Xyloglucan Endotransglycosylase Activity and Its Donor Substrate in the Elongation Zone of Arabidopsis Roots

We have developed a method for the colocalization of xyloglucan endotransglycosylase (XET) activity and the donor substrates to which it has access in situ and in vivo. Sulforhodamine conjugates of xyloglucan oligosaccharides (XGO–SRs), infiltrated into the tissue, act as acceptor substrate for the enzyme; endogenous xyloglucan acts as donor substrate. Incorporation of the XGO–SRs into polymeric products in the cell wall yields an orange fluorescence indicative of the simultaneous colocalization, in the same compartment, of active XET and donor xyloglucan chains. The method is specific for XET, as shown by competition experiments with nonfluorescent acceptor oligosaccharides, by negligible reaction with cello-oligosaccharide–SR conjugates that are not XET acceptor substrates, by heat lability, and by pH optimum. Thin-layer chromatographic analysis of remaining unincorporated XGO–SRs showed that these substrates are not extensively hydrolyzed during the assays. A characteristic distribution pattern was found in Arabidopsis and tobacco roots: in both species, fluorescence was most prominent in the cell elongation zone of the root. Proposed roles of XET that include cell wall loosening and integration of newly synthesized xyloglucans could thus be supported.

Vitamin C degradation in plant cells via enzymatic hydrolysis of 4-O-oxalyl-L-threonate

Increasing the l-ascorbate (vitamin C) content of crops could in principle involve promoting its biosynthesis or inhibiting its degradation. Recent progress has revealed biosynthetic pathways for ascorbate, but the degradative pathways remain unclear. The elucidation of such pathways could promote an understanding of the roles of ascorbate in plants, and especially of the intriguing positive correlation between growth rate and ascorbate oxidase (or its products). In some plants (Vitaceae), ascorbate is degraded via l-idonate to l-threarate (l-tartrate), with the latter arising from carbons 1–4 of ascorbate. In most plants, however (including Vitaceae), ascorbate degradation can occur via dehydroascorbate, yielding oxalate plus l-threonate, with the latter from carbons 3–6 of ascorbate. The metabolic steps between ascorbate and oxalate/l-threonate, and their subcellular location, were unknown. Here we show that this pathway operates extracellularly in cultured Rosa cells, proceeds via several novel intermediates including 4-O-oxalyl-l-threonate, and involves at least one new enzyme activity. The pathway can also operate non-enzymatically, potentially accounting for vitamin losses during cooking. Several steps in the pathway may generate peroxide; this may contribute to the role of ascorbate as a pro-oxidant that is potentially capable of loosening the plant cell wall and/or triggering an oxidative burst.

Phenolic Components of the Plant Cell Wall

Publisher Summary This chapter focuses on phenolic components of the plant cell wall. Phenol is the structural component common to all phenolic compounds. Both lignin and suberin are phenolic, or phenol-rich polymers, and their role in the life of the plant is well established. The chapter discusses lignin and phenolic acids in detail. There is also a description of other phenolic components in the plant cell wall such as cutin and suberin, which are lipophilic polymers. Sporopollenin is a highly resistant polymer found in the outer pollen wall of higher plants. Lignin is often present in the primary cell walls of fibers, xylem vessels, and tracheids. Lignin can be stained cytochemically by using a range of relatively specific reagents, including phloroglucinol-HCl, which responds to the cinnamaldehyde groups present in lignin. Phenolic compounds can also be detected in situ by their autofluorescence under ultraviolet light. During development, lignification often begins in the middle lamellae and primary cell walls and only later spreads into the secondary wall layers; in other tissues, however, only the secondary walls lignify. The lignin content of tissues can change quantitatively and qualitatively in response to various stimuli.

Intraprotoplasmic and wall-localised formation of arabinoxylan-bound diferulates and larger ferulate coupling-products in maize cell-suspension cultures

Abstract. Maize (Zea mays L.) cell cultures incorporated radioactivity from [14C]cinnamate into hydroxycinnamoyl-CoA derivatives and then into polysaccharide-bound feruloyl residues. Within 5–20 min, the CoA pool had lost its 14C by turnover and little or no further incorporation into polysaccharides then occurred. The system was thus effectively a pulse–chase experiment. Kinetics of radiolabelling of diferulates (also known as dehydrodiferulates) varied with culture age. In young (1–3 d) cultures, polysaccharide-bound [14C]feruloyl- and [14C]diferuloyl residues were both detectable within 1 min of [14C]cinnamate feeding. Thus, feruloyl residues were dimerised <1 min after their attachment to polysaccharides. For at least the first 2.3 h after [14C]cinnamate feeding, polysaccharide-bound [14C]diferuloyl residues remained almost constant at ≈7% of the total polysaccharide-bound [14C]ferulate derivatives. Since feruloyl residues are attached to polysaccharides <1 min after the biosynthesis of the latter, and >10 min before secretion, the data show that extensive feruloyl coupling occurred intra-protoplasmically. Exogenous H2O2 (1 mM) caused little additional feruloyl coupling; therefore, wall-localised coupling may have been peroxidase-limited. In older (e.g. 4 d) cultures, less intraprotoplasmic coupling occurred: during the first 2.5 h, polysaccharide-bound [14C]diferuloyl residues were a steady 1.4% of the total polysaccharide-bound [14C]ferulate derivatives. In contrast to the situation in younger cultures, exogenous H2O2 induced a rapid 4- to 6-fold increase in all coupling products, indicating that coupling in the walls was H2O2-limited. In both 2- and 4-d-old cultures, polysaccharide-bound 14C-trimers and larger coupling products exceeded [14C]diferulates 3- to 4-fold, but followed similar kinetics. Thus, although all known dimers of ferulate can now be individually quantified, it appears to be trimers and larger products that make the major contribution to cross-linking of wall polysaccharides in cultured maize cells. We argue that feruloyl arabinoxylans that are cross-linked before and after secretion are likely to loosen and tighten the cell wall, respectively. The consequences for the control of cell expansion and for the response of cell walls to an oxidative burst are discussed.

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.

Ultraviolet radiation drives methane emissions from terrestrial plant pectins

Recent studies demonstrating an in situ formation of methane (CH(4)) within foliage and separate observations that soil-derived CH(4) can be released from the stems of trees have continued the debate about the role of vegetation in CH(4) emissions to the atmosphere. Here, a study of the role of ultraviolet (UV) radiation in the formation of CH(4) and other trace gases from plant pectins in vitro and from leaves of tobacco (Nicotiana tabacum) in planta is reported. Plant pectins were investigated for CH(4 )production under UV irradiation before and after de-methylesterification and with and without the singlet oxygen scavenger 1,4-diazabicyclo[2.2.2]octane (DABCO). Leaves of tobacco were also investigated under UV irradiation and following leaf infiltration with the singlet oxygen generator rose bengal or the bacterial pathogen Pseudomonas syringae. Results demonstrated production of CH(4), ethane and ethylene from pectins and from tobacco leaves following all treatments, that methyl-ester groups of pectin are a source of CH(4), and that reactive oxygen species (ROS) arising from environmental stresses have a potential role in mechanisms of CH(4) formation. Rates of CH(4 )production were lower than those previously reported for intact plants in sunlight but the results clearly show that foliage can emit CH(4) under aerobic conditions.

Mixed‐linkage (1→3,1→4)‐β‐d‐glucan is a major hemicellulose of Equisetum (horsetail) cell walls

Mixed-linkage (1-->3,1-->4)-beta-d-glucan (MLG) is a hemicellulose reputedly confined to certain Poales. Here, the taxonomic distribution of MLG, and xyloglucan, especially in early-diverging pteridophytes, has been re-investigated. Polysaccharides were digested with lichenase and xyloglucan endoglucanase (XEG), which specifically hydrolyse MLG and xyloglucan, respectively. The oligosaccharides produced were analysed by thin-layer chromatography (TLC), high-pressure liquid chromatography (HPLC) and alkaline peeling. Lichenase yielded oligo-beta-glucans from all Equisetum species tested (Equisetum arvense, Equisetum fluviatile, Equisetum scirpoides, Equisetum sylvaticum and Equisetum xtrachyodon). The major product was the tetrasaccharide beta-glucosyl-(1-->4)-beta-glucosyl-(1-->4)-beta-glucosyl-(1-->3)-glucose (G4G4G3G), which was converted to cellotriose by alkali, confirming its structure. Minor products included G3G, G4G3G and a nonasaccharide. By contrast, poalean MLGs yielded G4G3G > G4G4G3G > nonasaccharide > dodecasaccharide. No other pteridophytes tested contained MLG, including Psilotum and eusporangiate ferns. No MLG was found in lycopodiophytes, bryophytes, Chara or Nitella. XEG digestion showed that Equisetum xyloglucan has unusual repeat units. Equisetum, an exceedingly isolated genus whose closest living relatives diverged > 380 million years ago, has evolved MLG independently of the Poales. Equisetum and poalean MLGs share basic structural motifs but also exhibit clear-cut differences. Equisetum MLG is firmly wall-bound, and may tether neighbouring microfibrils. It is also suggested that MLG acts as a template for silica deposition, characteristic of grasses and horsetails.

Inhibition of auxin-stimulated growth of pea stem segments by a specific nonasaccharide of xyloglucan

Hemicellulose extracted from cell walls of suspension-cultured rose (Rosa “Pauls Scarlet”) cells was digested with cellulase from Trichoderma viride. The quantitatively major oligosaccharide products, a nonasaccharide and a heptasaccharide derived from xyloglucan, were purified by gel permeation chromatography. The nonasaccharide was found to inhibit the 2,4-dichlorophenoxy-acetic-acid-induced elongation of etiolated pea (Pisum sativum) stem segments. This confirms an earlier report (York et al., 1984, Plant Physiol. 75, 295–297). The inhibition of elongation by the nonasaccharide showed a maximum at around 10-9M with higher and lower concentrations being less effective. The heptasaccharide did not significantly inhibit elongation at 10-7–10-10M and also did not affect the inhibition caused by the nonasaccharide when co-incubated with the latter.