STUDIES ON PLANT GROWTH-REGULATING SUBSTANCES

Fifteen series of aryloxyacetic, α-aryloxy-propionic, -butyric and -isobutyric acids and the corresponding arylthio-analogues have been synthesized and their growth-regulating activity has been assessed in the tomato-leaf epinasty, wheat cylinder and the pea curvature tests. It is shown that in general arylthio-derivatives are less active than their aryloxy-analogues. Although for a cell elongation response the presence in the molecule of an a-hydrogen atom is usually necessary, three exceptions to this generalization have now been found. A lower activity in the 3:5-dichloro- compared with the 2:6-dichloro-compounds has been demonstrated. Evidence is presented to show that an ortho hydrogen atom in the nucleus is not an essential requirement for cell elongating activity as is postulated by other workers.     

STUDIES ON PLANT GROWTH-REGULATING SUBSTANCES

Three synthetic growth-regulating substances, α-(2-naphthoxy)-, α-(2:4-dichloro-phenoxy)- and α-(2:4:5-trichlorophenoxy)-propionic acids, have been resolved into their (+)- and (-)-forms. The physiological activity of these isomers and of the racemic compounds has been investigated using the pea curvature and Avena cylinder test methods of assessment.
The (+)-isomers in each case displayed high activity in both tests, whereas that exhibited by the (-)-isomers was negligible. The racemic compounds showed intermediate activity.
The mode of action of aryloxyalkylcarboxylic acids is discussed in the light of the results presented. These considerations suggest the possibility that the activity of the (+)-isomers might be antagonized by the presence of the corresponding inactive enantiomorphs. Our experiments show that such antagonism does in fact operate.     

STUDIES ON PLANT GROWTH-REGULATING SUBSTANCES

A study has been made of the growth-regulating activity of a number of phenoxy acids possessing halogen or methyl substituents in the 2:6- or 33- positions of the nucleus. The pea curvature, Avena cylinder elongation and tomato-leaf epinasty tests have been used for assessing biological activity. The results show that in general 3:s- is more closely associated with inactivity than 2:6- substitution. The introduction of a further halogen atom into the 4- position does not enhance activity in the 2:6- compounds but does so in the 3:s- derivatives. Although the 2:4:6-trichloro- and 2:4:6-tribromo-phenoxyacetic acids are almost inactive, a:4-dichloro-6-fluoro- and 2:4-dibromo-6-fluoro- phenoxyacetic acids are very highly active. When a methyl group is substituted into the side-chain of certain phenoxyacetic acids possessing 2:6- substituents, growth-promoting activity is increased. Some of these findings conflict with recent theories on mode of action of phenoxy acids which are discussed in relation to the present work.     

Studies on plant growth-regulating substances XXVIII. Halogen-substituted benzoic acids

The plant growth-regulating activities of chloro-, bromo- and iodo-isomers in 2,5-, 2,3,5- and 2,3,6-substituted benzoic acids were assessed in the wheat cylinder, pea segment and pea curvature tests. Their effects in the tomato-leaf epinasty test were also investigated. Replacing an ortho-chloro atom by bromine had little or no effect on activity. An ortho-iodo substituent, however, reduced activity in the 2,3,6-substituted series and almost completely inhibited it in the 2,5- substituted series. This decreased activity of ortho-iodo-substituted isomers is visualized as being due to a steric inhibition of the attachment of the carboxyl group to its receptor unit prior to the initiation of the growth response.     

Studies on plant growth-regulating substances. XXX. The plant growth-regulating activity of substituted phenols

The growth-regulating activity of a number of substituted monophenols and related compounds has been assessed in the wheat cylinder, pea segment, pea curvature and tomato-leaf epinasty tests and the effect on activity of position, size and electronic nature of the substituents has been studied. The results indicate that, for high auxin activity, electron-attracting substituents, having certain steric properties must be substituted in the 2- and 6-positions. Furthermore, at least one of these ortho-substituents must be capable of intramolecular bonding with the hydrogen of the phenolic hydroxyl group. Any substituent in the para-position to the hydroxyl group leads to complete loss of activity and activity is reduced by meta-substitution. This new group of phenolic plant growth regulators is considered in relation to other synthetic auxins from the viewpoints of structural requirements for activity and current theories on mode of action.     

Studies on plant growth-regulating substances

The metabolism of certain 2,6-disubstituted phenols that possess high auxin activity in the pea segment, pea curvature and tomato-leaf epinasty tests, but are much less active in the wheat cylinder test, has been investigated in wheat, pea and tomato tissue. Metabolites were identified by thin-layer chromatography and a semi-quantitative assay method was developed. The low activity of 2,6-dihalogenophenols and inactivity of 2-halogeno-6-nitro-phenols and 3-halogeno-2-hydroxybenzonitriles in the wheat cylinder test was caused by rapid metabolic conversion of the compounds in this tissue to inactive compounds by a process involving hydroxylation of the aromatic ring in the para- position. No such inactivation occurred in pea and tomato tissues. Evidence for a novel detoxification of nitrophenols within both pea and wheat tissue was obtained; 2-bromo–6-nitrophenol was converted via 2-bromo-6-aminophenol to N-acetyl-2-bromo-6-aminophenol. Certain 3-halogeno-2-hydroxybenzaldehydes and corresponding aceto-phenones, although fulfilling the necessary structural and electronic criteria for auxin activity, are inactive. Metabolic studies indicate that this is because they are metabolized in wheat, pea and tomato tissues to compounds not possessing the structural requirements for auxin activity.     

STUDIES ON PLANT GROWTH-REGULATING SUBSTANCES

The cress-root test and the maize-root segment tests, both of which depend on the measurement of root-growth inhibition, have been used to assess the growth-regulating activity of a wide range of phenoxyalkanecarboxylic acids. The results are presented and compared with those obtained with the same compounds in other tests in which shoot tissues were employed. The results are also discussed in relation to current theories on mode of action and structure/activity relationship. These studies included the 'α-hydrogen' effect, the activity of stereoisomers and activity of homologues of seventeen series of ω-phenoxyalkanecarboxylic acids, considered in relation to their degradation within the tissues by β-oxidation. Earlier findings were largely confirmed. Compounds which had previously been shown to be active in shoot tests such as the wheat-cylinder, the pea-curvature and the pea-segment tests, were found to be active in the root-growth inhibition tests and, furthermore, the degree of activity was usually similar.
It is concluded that the two root tests investigated are of real value in assessing the activity of phenoxy acids in studies on the relationships between chemical structure and plant growth-regulating activity.     

Stereospecificity of NADH-ferricyanide reductase is a convenient marker for the endoplasmic reticulum of plant cells

The stereospecificity of NADH-ferricyanide reductase and NADH-cytochrome c reductase in the endoplasmic reticulum (ER) for the α-hydrogen on the nicotinamide ring is presented as a very sensitive and convenient assay to detect ER contamination in preparations of membranes lacking α-specific NADH-acceptor reductase, such as the plasma membrane and the tonoplast. The experimental details of the assay are given and the limitations explored (time-course, amount of protein, possible side reactions, speed, reproducibility, etc.). The NADH-ferricyanide reductase activity of plasma membranes from spinach and sugarbeet leaf was completely β-specific and always showed a latency (increase upon addition of Triton X-100), whereas the α-specificity in the ER was non-latent. This is consistent with the presence of mainly right-side-out vesicles in preparations of plasma membranes with the binding site for NADH and ferricyanide on the inner, cytoplasmic surface. In contrast, right-side-out ER vesicles have the binding site on the outer, cytoplasmic surface. The addition of as little as 1% of the α-specific ER (on an NADH-ferricyanide activity basis) to the spinach leaf plasma membrane could be detected with the stereospecificity assay. Wheat root plasma membrane showed some α-specificity (in addition to β-specificity) which was probably due to ER contamination since the activity was non-latent. The stereospecificity assay is also shown to be useful in monitoring the separation of tonoplast vesicles from ER vesicles by countercurrent distribution of a light microsomal fraction. It follows that the NADH-acceptor reductase activities in preparations of plasma membrane and tonoplast are due to distinct enzymes characteristic for those membranes.     

STUDIES ON PLANT GROWTH-REGULATING SUBSTANCES XI. AUXIN ANTAGONISM IN RELATION TO A THEORY ON MODE OF ACTION OF ARYL- AND ARYLOXY-ALKANECARBOXYLIC ACIDS

A study has been made of the antagonistic effects of a range of phenoxy and other acids on the growth-promoting activity of a number of auxins as assessed by the Avena cylinder and pea curvature tests. The compounds examined as inhibitors were selected on a logical basis, these molecules not possessing the structural requirements considered necessary for growth-regulating activity, yet possessing groupings which might allow them to accumulate at a site of action from which the growth response by auxins might be initiated. Our previous demonstrations that inactive stereoisomers of x-aryloxy propionic acids can competitively inhibit the activity of their active enantiomorph has been confirmed and a similar antagonism has been found to operate with other inactive aryloxy acids. Such competitive antagonism is discussed in relation to a 'three-point' contact theory on mode of action.     

Studies on plant growth-regulating substances. XLIII. 2 -Chloro -3 -phenoxypropionitriles

A number of ring substituted 2-chloro-3-phenoxypropionitriles have been prepared and their plant growth-regulating activity has been assessed in the wheat coleoptile, pea segment and pea curvature tests, and also by spray treatments on tomato and dwarf French bean plants. The activities of these compounds are compared with those of previously studied 2-chloro-3-phenylpropionitriles and the results are discussed in terms of chemical structure/biological activity relationships and mode of action.     

Studies on plant growth-regulating substances: XXXIII. The influence of ring substituents on the plant growth-regulating activity of phenylacetic acid

A comprehensive range of phenylacetic acids substituted with nitro, halogen, methyl, amino, hydroxyl and N-acetylamino groups have been synthesized and their growth-regulating activities assessed in the wheat cylinder, pea curvature and pea segment tests. The influence of substituents on molecular shape is shown to be more important in determining activity than their effects on electron distribution. Studies with 2,6-disubstituted phenylacetic acids have indicated that the most active compounds can attain a certain spatial configuration in which one surface of the molecule, including the plane of the ring system, is flat and the carboxyl group is above with its axis of rotation perpendicular to this surface. Positional requirements for growth-regulating activity in phenylacetic acids are shown to be less important than in the phenoxyacetic and benzoic acids.     

Studies on plant growth-regulating substances

Series of I, I-dichloro-2-phenoxyethanes, α,α-dichlorotoluenes and I, I-dihalo-2-phenylethanes have been examined for plant growth-regulating activity in a number of tests. Some compounds were shown to be active and the results, together with those obtained from metabolism studies, are discussed in terms of chemical structure/biological activity relationships and mode of action.     

Phytotoxic effects of Sicyos deppei (Cucurbitaceae) in germinating tomato seeds

The phytotoxic effect of allelochemicals is referred to as allelochemical stress and it is considered a biotic stress. Sicyos deppei G. Don (Cucurbitaceae) is an allelopathic weed that causes phytotoxicity in Lycopersicon esculentum , delaying seed germination and severely inhibiting radicle growth. This paper reports in in vitro conditions, the effects of the aqueous leachate of S. deppei —throughout tomato germination times—on (1) the dynamics of starch and sugars metabolism, (2) activity and expression of the cell wall enzymes involved in endosperm weakening that allows the protrusion of the radicle, and (3) whether abscisic acid (ABA) is involved in this altered metabolic processes. Results showed that S. deppei leachate on tomato seed germination mainly caused: (1) delay in starch degradation as well as in sucrose hydrolysis; (2) lower activity of sucrose phosphate synthase, cell wall invertase, and α-amylase; being sucrose phosphate synthase (SPS) gene expression down-regulated, and the last two up regulated; (3) also, lower activity of endo β-mannanase, β-1,3 glucanase, α-galactosidase, and exo-polygalacturonase with altered gene expression; and (4) higher content of ABA during all times of germination. The phytotoxic effect of S. deppei aqueous leachate is because of the sum of many metabolic processes affected during tomato seed germination that finally is evidenced by a strong inhibition of radicle growth.     

Studies on plant growth-regulating substances XXV. The plant growth-regulating activity of cinnamic acids

Effects of ring substitution on the plant growth-regulating activities of trans- and cis-cinnamic acids have been investigated in the wheat cylinder, pea segment and pea curvature tests. Most of the cis- acids were shown to be active. Substitution of fluorine, chlorine or bromine into the ring of cis-cinnamic acid in most cases increased the activity. The results are discussed in relation to mode of action and chemical structure/biological activity relationships: 4-chlorobenzoic acid is shown to act as a competitive antagonist towards 4-chloro-cis-cinnamic acid.     

<Emphasis Type="Italic">Xanthomonas gardneri</Emphasis> exoenzymatic activity towards plant tissue

Bacterial spot caused by Xanthomonas spp. is an important tomato and pepper disease worldwide. Recent outbreaks of bacterial spot disease in Central Brazil and Canada have been attributed to Xanthomonas gardneri, which is also recognized as group D of Xanthomonas campestris pv. vesicatoria. Carotenoid-like pigments called xanthomonadins, which are diagnostic for yellow Xanthomonas spp., were extracted from X. gardneri. It was shown that the model plant Arabidopsis thaliana, member of the Brassicaceae family, can develop disease symptoms in response to different isolates of X. gardneri. Secretion of enzymes has been shown to play an important role in pathogenicity for different pathogens, and to begin to understand the interaction of X. gardneri and A. thaliana, a biochemical analysis of secreted proteins in the presence of A. thaliana leaves was performed. Different enzymatic activities such as for cellulase, α-arabinofuranosidase, pectinase, invertase and xylanase were assayed. In the presence of leaves, cellulase activity was highest after 60 and 72 h of growth and α-arabinofuranosidase activity was detected between 12 and 72 h of growth. Pectinase, invertase and xylanase activities were not detected. Cellulase and α-arabinofuranosidase activities may be important for X. gardneri acquisition of plant nutrients through degradation of cellulose fibers and hemicellulose of the cell wall, respectively, to the invasion of the host tissue and/or may generate signal molecules that are recognized by the plant. This is the first study to address how X. gardneri responds to host plant tissue.     

STUDIES ON PLANT GROWTH-REGULATING SUBSTANCES

A number of polycyclic carboxylic acids have been prepared and their plant growth-regulating activity has been assessed in the wheat-cylinder, pea-curvature and pea-segment tests. The compounds were selected in relation to a theory on the mode of action of plant-growth substances which involves the association of three essential groupings in the molecule with specifically sited receptor centres within the plant cell. High activity was found only in those compounds possessing a > CHCOOH grouping adjacent to an aromatic ring. It is concluded that molecular size, stereochemical and electronic considerations may all operate in determining whether compounds of this type show activity. The results obtained together with those of other workers are discussed in relation to current theories on mode of action.     

Relationship between alpha synuclein phosphorylation, proteasomal inhibition and cell death: relevance to Parkinson's disease pathogenesis

Alpha synuclein can be phosphorylated at serine129 (P-S129), and the presence of highly phosphorylated α-synuclein in Lewy bodies suggests changes to its phosphorylation status has an important pathological role. We demonstrate that the kinase(s) responsible for α-synuclein S129 phosphorylation is constitutively active in SH-SY5Y cells and involves casein kinase 2 activity. Increased oxidative stress or proteasomal inhibition caused significant elevation of P-S129 α-synuclein levels. Under these conditions, similar increases in P-S129 α-synuclein were found in both sodium dodecyl sulphate lysates and Triton extracts indicating the phosphorylated protein was soluble and did not lead to aggregation. The rate of S129 phosphorylation was increased in response to proteasomal inhibition indicating a higher activity of the relevant kinase. Cells expressing the phosphorylation mimic, S129D α-synuclein increased cell death and enhanced sensitivity to epoxomycin exposure. Proteasomal inhibition markedly decreased S129D α-synuclein turnover suggesting proteasomal inhibition leads to the accumulation of P-S129 α-synuclein through an increase in the kinase activity and a decrease in protein turnover resulting in increased cell death. We conclude that S129 phosphorylation is toxic to dopaminergic cells and both the levels of S129 phosphorylated protein and its toxicity are increased with proteasomal inhibition emphasising the interdependence of these pathways in Parkinson's disease pathogenesis.     

Characterization of phytochelatin synthase from tomato

The enzyme that synthesizes Cd-binding phytochelatins (PCs), PC synthase, has been studied in tomato ( Lycopersicon esculentum ) cell cultures and plants. This enzyme transfers γ-GluCys from GSH or PC to either GSH or an existing polymer of (γ-GluCys)_nGly. PC synthase from tomato requires GSH or PCs as substrates but cannot utilise γ-GluCys or GSSG. PC synthase is activated both in vivo and in vitro by a variety of heavy metal ions, including Cd²⁺, Ag⁺, Cu²⁺, Au⁺, Zn²⁺, Fe²⁺, Hg²⁺ and Pb²⁺. In crude protein extracts from tomato cells the enzyme has an apparent K_m of 7.7 m M for GSH in the presence of 0.5 m M Cd²⁺, and exhibits maximum activity at pH 8.0 and 35°C. PC synthase is present in tomato cells grown in the absence of Cd. The level of enzyme activity is regulated during the cell culture cycle, with the highest activity occurring 3 days after subculture. Cadmium-resistant tomato cells growing in medium containing 6 m M CdCl₂ have a 65% increase in PC synthase activity compared to unselected cells. PC synthase is also present in roots and stems of tomato plants, but not in leaves or fruits. The distribution of the enzyme in tomato plants and regulation of PC synthase activity in tomato cells indicate that PC synthase, and PCs, may have additional functions in plant metabolism that are not directly related to the formation of Cd-PC complexes in response to cadmium.