M. A. Hall

The polysaccharide structure of potato cell walls: Chemical fractionation

Cell walls of potato tubers were fractionated by successive extraction with various reagents. A slightly degraded pectic fraction with 77% galacturonic acid was extracted in hot, oxalate-citrate buffer at pH 4. A further, major pectic fraction with 38% galacturonic acid was extracted in cold 0.1 M Na2CO3 with little apparent degradation. These two pectic fractions together made up 52% of the cell wall. Most of the oxalate-citrate fraction could alternatively be extracted with cold acetate-N,N′,N′-tetracetic acid (CDTA) buffer, a non-degradative extractant which nevertheless removed essentially all the calcium ions. This fraction was therefore probably held only by calcium binding, and the remainder of the pectins by covalent bonds. Electrophoresis showed that both pectic fractions contained a range of molecular types differing in composition, with a high arabinose: galactose ratio as well as much galacturonic acid in the most extractable fractions. From methylation data, the main side-chains were 1,4′-linked galactans and 1,5′-linked arabinans, with smaller quantities of covalently attached xyloglucan. Extraction with NaOH-borate removed a small hemicellulose fraction and some cellulose. The main hemicelluloses were apparently a galactoxyloglucan, a mannan or glucomannan and an arabinogalactan.

Plant growth regulators II: Cytokinins, their analogues and antagonists.

Hormones in plants differ from most of those in animals by having pleiotropic effects; that is, they are involved in the control of a wide range of developmental processes. At the same time the effect of a hormone on any developmental process depends on the species. For example, ethylene inhibits growth in dicotyledons and most monocotyledons but is promotory in deepwater rice and other hydrophytes. Moreover, two or more hormones can interact synergistically or antagonistically in many circumstances. Equally, any given hormone may affect the biosynthesis or metabolism of another, thus affecting endogenous levels. The issue is further complicated by the fact that environmental factors e.g. light, water status, wounding, pathogens may modify responses and indeed hormone levels themselves. The reason for this appears to be that hormones (and growth regulators) and environmental factors share many components in their transduction chains (i.e. the very early events which occur after the signal abiotic or biotic is perceived by the plant tissue). These transduction chains interact to produce an integrated response.

Nitric Oxide Interacts with Salicylate to Regulate Biphasic Ethylene Production during the Hypersensitive Response

C2H4 is associated with plant defense, but its role during the hypersensitive response (HR) remains largely uncharacterized. C2H4 production in tobacco (Nicotiana tabacum) following inoculation with HR-eliciting Pseudomonas syringae pathovars measured by laser photoacoustic detection was biphasic. A first transient rise (C2H4-I) occurred 1 to 4 h following inoculation with HR-eliciting, disease-forming, and nonpathogenic strains and also with flagellin (flg22). A second (avirulence-dependent) rise, at approximately 6 h (C2H4-II), was only seen with HR-eliciting strains. Tobacco leaves treated with the C2H4 biosynthesis inhibitor, aminoethoxyvinylglycine, suggested that C2H4 influenced the kinetics of a HR. Challenging salicylate hydroxylase-expressing tobacco lines and tissues exhibiting systemic acquired resistance suggested that C2H4 production was influenced by salicylic acid (SA). Disrupted expression of a C2H4 biosynthesis gene in salicylate hydroxylase tobacco plants implicated transcriptional control as a mechanism through which SA regulates C2H4 production. Treating leaves to increase oxidative stress or injecting with SA initiated monophasic C2H4 generation, but the nitric oxide (NO) donor sodium nitroprusside initiated biphasic rises. To test whether NO influenced biphasic C2H4 production during the HR, the NO synthase inhibitor NG-nitro-l-arginine methyl ester was coinoculated with the avirulent strain of P. syringae pv phaseolicola into tobacco leaves. The first transient C2H4 rise appeared to be unaffected by NG-nitro-l-arginine methyl ester, but the second rise was reduced. These data suggest that NO and SA are required to generate the biphasic pattern of C2H4 production during the HR and may influence the kinetics of HR formation.

Studies on ethylene binding by cell free preparations from cotyledons of Phaseolus vulgaris L. : II. Effects of structural analogues of ethylene and of inhibitors.

Various protein reactive agents such as dithioerythritol, dithiothreitol, mercaptoethanol and p-chloromercuribenzoate inhibit binding of ethylene to cell free preparations of Phaseolus vulgaris L. The effect of the thiols is partially reversed by treatment with diamide; occupation of the binding site by ligand diminishes the inhibition caused by p-chloromercuribenzoate but not that caused by thiols. Growth regulators other than ethylene do not affect binding. Physiologically active structural analogues of ethylene competitively inhibit binding of the growth regulator and their relative effectiveness in the cell free system closely resembles that in developmental processes controlled ethylene.

Explanatory Analysis of the Metabolome Using Genetic Programming of Simple, Interpretable Rules

Genetic programming, in conjunction with advanced analytical instruments, is a novel tool for the investigation of complex biological systems at the whole-tissue level. In this study, samples from tomato fruit grown hydroponically under both high- and low-salt conditions were analysed using Fourier-transform infrared spectroscopy (FTIR), with the aim of identifying spectral and biochemical features linked to salinity in the growth environment. FTIR spectra of whole tissue extracts are not amenable to direct visual analysis, so numerical modelling methods were used to generate models capable of classifying the samples based on their spectral characteristics. Genetic programming (GP) provided models with a better prediction accuracy to the conventional data modelling methods used, whilst being much easier to interpret in terms of the variables used. Examination of the GP-derived models showed that there were a small number of spectral regions that were consistently being used. In particular, the spectral region containing absorbances potentially due to a cyanide/nitrile functional group was identified as discriminatory. The explanatory power of the GP models enabled a chemical interpretation of the biochemical differences to be proposed. The combination of FTIR and GP is therefore a powerful and novel analytical tool that, in this study, improves our understanding of the biochemistry of salt tolerance in tomato plants.

Studies on ethylene binding by cell-free preparations from cotyledons of Phaseolus vulgaris L. : I. Separation and characterisation.

The preparation is described of a cell-free system from developing cotyledons of Phaseolus vulgaris cv. Canadian Wonder which is capable of binding ethylene. The binding is saturable and the apparent dissociation constant for ethylene is 6.4·10-10 M in solution. The binding site is associated with subcellular particles and treatment with Triton X-100 results in substantial solubilisation of the activity. The kinetics of association and dissociation of the ligand and the binding site are described. The system is heat labile and binding activity is diminished by treatment with some proteolytic enzymes.

The effect of ethylene on MAPKinase-like activity in Arabidopsis thaliana

Protein kinase activity was studied in cytosolic extracts from leaves of wild type Arabidopsis thaliana, the ethylene‐insensitive mutant, etr1, and the constitutive triple‐response mutant, ctr1. Treatment of wild type with ethylene resulted in increased myelin basic protein (MBP) phosphorylation. In etr1, constitutive protein kinase activity was lower than in wild type, but in ctr1, activity was enhanced. A protein of M r∼47 kDa associated with MBP‐phosphorylating activity was detected using in gel protein kinase assays and phosphorylation of this protein was promoted by ethylene treatment in wild type while activity in the mutants reflected that of MBP phosphorylation. Both MAPKinase (ERK 1) and phosphotyrosine antibodies immunoprecipitated MBP‐phosphorylating activity and detected a polypeptide band at M r∼47 kDa. Immunoprecipitated MBP‐phosphorylating activity was again much lower in etr1 compared to wild type but much higher in ctr1. Antibodies to phosphorylated MAPKinase recognised proteins at ∼47 kDa and the signal was upregulated in response to ethylene. The data obtained suggest that the detected protein(s) is a MAPKinase and provide further evidence confirming that a MAPKinase cascade(s) is involved in ethylene signal transduction.

The compartmentation of ethylene in developing cotyledons of Phaseolus vulgaris L.

Isolated cotyledons of Phaseolus vulgaris L. cv. Canadian Wonder accumulated 14C2H4 (0.7–1 μl l-1) from air to give partition coefficients of 1 to 4, which greatly exceeded the value obtained with steam killed cotyledons (0.05) and with water (0.11). After 14C2H4 treatment, 98% of the 14C in the tissue remained as 14C2H4. The labelled ethylene accumulated by cotyledons was released only slowly (1–10% h-1) either in an air stream or into toluene. Heating to 60°C for 2 h, but not freezing and thawing, caused the immediate release of 14C2H4 from the tissue. Propylene and vinyl chloride competitively inhibited the accumulation of 14C2H4.Cotyledons emanated endogenous ethylene at a very low rate but after heating (although not freezing and thawing) 13 nl of ethylene per g fresh mass were released within minutes. It was concluded that french bean cotyledons hold ethylene in a compartmented form in sufficient amount to account for at least 200 h of emanation.

Ethylene Regulates Monomeric GTP-Binding Protein Gene Expression and Activity in Arabidopsis

Ethylene rapidly and transiently up-regulates the activity of several monomeric GTP-binding proteins (monomeric G proteins) in leaves of Arabidopsis as determined by two-dimensional gel electrophoresis and autoradiographic analyses. The activation is suppressed by the receptor-directed inhibitor 1-methylcyclopropene. In theetr1-1 mutant, constitutive activity of all the monomeric G proteins activated by ethylene is down-regulated relative to wild type, and ethylene treatment has no effect on the levels of activity. Conversely, in the ctr1-1 mutant, several of the monomeric G proteins activated by ethylene are constitutively up-regulated. However, the activation profile of ctr1-1does not exactly mimic that of ethylene-treated wild type. Biochemical and molecular evidence suggested that some of these monomeric G proteins are of the Rab class. Expression of the genes for a number of monomeric G proteins in response to ethylene was investigated by reverse transcriptase-PCR. Rab8 andAra3 expression was increased within 10 min of ethylene treatment, although levels fell back significantly by 40 min. In theetr1-1 mutant, expression of Rab8 was lower than wild type and unaffected by ethylene; inctr1-1, expression of Rab8 was much higher than wild type and comparable with that seen in ethylene treatments. Expression in ctr1-1 was also unaffected by ethylene. Thus, the data indicate a role for monomeric G proteins in ethylene signal transduction.

The effect of ethylene and cytokinin on guanosine 5'-triphosphate binding and protein phosphorylation in leaves of Arabidopsis thaliana

Abstract. Binding of [α-32P]guanosine 5′-triphosphate ([α-32P]GTP) has been demonstrated in a Triton X-100-solubilised membrane fraction from leaves of Arabidopsis thaliana (L.) Heynh. Binding was stimulated by 1 h pre-treatment of leaves with ethylene and this effect was antagonised by the inclusion of N6-benzyladenine in the medium used for homogenisation. The ethylene-insensitive mutants eti5 and etr showed contrasting responses. In eti5 the constitutive level of GTP binding was higher than in the wild type whereas in etr the level was much lower. Neither ethylene nor cytokinin affected GTP binding in the mutants. The GTP-binding activity was localised in two bands at 22 and 25 kDa, both of which were immunoprecipitated by anti-pan-Ras antibodies, indicating that the activity is due to small GTP-binding proteins. In a similar membrane fraction, ethylene was shown to increase protein phosphorylation and benzyladenine antagonised this effect. In eti5 the constitutive level of protein phosphorylation was higher than in the wild type, but benzyladenine increased activity substantially while ethylene was without effect. In etr, protein phosphorylation was lower than in the wild type, ethylene was without effect, but cytokinin increased activity. A protein of Mr 17 kDa was detected on gels using antibodies to nucleoside diphosphate kinase. Phosphorylation of this protein was upregulated by ethylene but nucleoside diphosphate kinase activity was unaffected. The results are compared with the effect of the two hormones on the senescence of detached leaves and discussed in relation to pathways proposed for ethylene signal transduction.