Enzymatic production of the plant growth inhibitor,xanthoxin

Summary Incubation of violaxanthin with lipoxygenase and linoleate gave rise to the plant growth inhibitor, xanthoxin; and the yields were reduced to 1/10 and 1/20 by the omission of lipoxygenase and both lipoxygenase and linoleate respecively.     

Effects of norflurazon, an inhibitor of carotenogenesis, on abscisic acid and xanthoxin in the caps of gravistimulated maize roots

Maize seeds were germinated in the dark in the presence of the carotenoid synthesis inhibitor norflurazon and the teveis of abscisic acid, xanthoxin and total carotenoids were measured in the root cap and in the adjacent 1.5 mm segment. In norflurazon-treated roots abscisic acid levels were markedly reduced, but an increase occurred in the levels of xanthoxin, a compound structurally and physiologically similar to abscisic acid. In the cultivar of maize ( Zea mays L. cv. Merit) used for this work, brief illumination of the root is required for gravitropic curving. Following illumination both control and norflurazon-treated roots showed normal gravitropic curvature, however, the rate of curvature was delayed in norflurazon-treated roots. Our data from norflurazon-treated roots are consistent with a role for xanthoxin in maize root gravitropism. The increase in xanthoxin in the presence of an inhibitor of carotenoid synthesis suggests that xanthoxin and abscisic acid originate, at least in part, via different metabolic pathways.     

Relationships between xanthoxin,phototropism, and elongation growth in the sunflower seedling Helianthus annuus L.

For phototropic curvature of a green sunflower seedling, only the hypocotyl has to be illuminated; the tip and cotyledons are not involved in stimulus perception. The etiolated seedling is phototropically insensitive, illumination of only the hypocotyl renders it sensitive. It is concluded that the photoreceptor is located within the responding organ. In curving seedlings, the endogenous indoleacetic acid (IAA) remains evenly distributed. However, the inhibitor, xanthoxin (Xa), accumulates on the illuminated side. The degree of phototropic response is generally related to the concentration of Xa. The amount of phototropic curvature is independent of the rate of elongation growth, the former can be changed without affecting the latter, and vice versa. The data conflict with the Cholodny-Went theory, whereas they support the hypothesis of Blaauw that the phototropic reaction is caused by the local accumulation of a growth-inhibiting substance on the irradiated side.Abbreviations CCC chlormequat, (2-chloroethyl)trimethylammonium chloride - GA₃ gibberellic acid - IAA indole-3-acetic acid - Xa xanthoxin     

Abscisic acid physiology and biosynthesis in higher plants

Abscisic acid (ABA) has been postulated to modulate several aspects of plant growth and development. While it is tempting to attribute changes in growth and development to a specific hormone such as ABA, the reality is that these processes are complex and poorly understood. Since there is so little known about basic biochemical events that occur during growth and development, it is difficult ot unambiguously assign a role for ABA in any process. Becuse of this, many of the cited effects of ABA on growth and development have not been conclusively demonstrated. Howver, it is clear that ABA has a function in ameliorating water-stress and preventing vivipary. The roles of ABA in bud dormancy and growth still remain unclear. With the use of biosynthesis inhibitors and mutants which block ABA accumulation, it has been shown that ABA does not play a role in gravitropism.
Knowledge of how the levels of any particular growth regulator are modulated is essential for the understanding of its physiology. The use of mutants, inhibitors and heavy isotopes suggests that ABA may be derived from a carotenoid rather than directly from farnesyl pyrophosphate (FPP), and that the cleavage of a carotenoid is the rate limiting step. However, the relative contribution of each pathway (and the role of xanthoxin) in ABA biosynthesis remain unknown.     

The role of xanthoxin in the inhibition of pea seedling growth by red light

Intermittent periods of red light illumination inhibit the growth of dwarf pea seedlings within 1 day and stop length increase completely in 3 days. Cis,trans-xanthoxin is synthesized within the plant during the 1st day of illumination and reaches a maximum level on the 3rd day. The red light treatment also causes an increase in the levels of violaxanthin, linoleic acid and peroxidase, lipoxygenase and carotene-bleaching activities in the plant. The possible control of xanthoxin production is discussed.     

Azospirillum sp. Promotes Root Hair Development in Tomato Plants through a Mechanism that Involves Ethylene

Tomato seeds were inoculated with the plant growth–promoting rhizobacteria Azospirillum brasilense FT326, and changes in parameters associated with plant growth were evaluated 15 days after inoculation. Azospirilla were localized on roots and within xylematic tissue. An increase in shoot and root fresh weight, main root hair length, and root surface indicated that inoculation with A. brasilense FT 326 resulted in plant growth improvement. The levels of indole-3-acetic acid (IAA) and ethylene, two of the phytohormones related to plant growth, were higher in inoculated plants. Exogenously supplied ethylene mimicked the effect of inoculation, and the addition of an inhibitor of its synthesis or of its physiological activity completely blocked A. brasilense growth promotion. Based on our results, we propose that the process of growth promotion triggered by A. brasilense inoculation involves a signaling pathway that has ethylene as a central, positive regulator.     

Hairy root and tissue cultures of <Emphasis Type="Italic">Leucojum aestivum</Emphasis> L.—relationships to galanthamine content

Galanthamine, an isoquinoline alkaloid acetylcholinesterase inhibitor, is an important agent used all around the world for the symptomatic treatment of senile dementia of the Alzheimer’s type. The production of this metabolite and the availability of the plant are limited and prompted the search for an alternative way to obtain this valuable metabolite using in vitro cultures of Leucojum aestivum L. It is known that cell differentiation level shows a major influence upon the accumulation of alkaloids. For this reason, tissue cultures of L. aestivum showing different stages of morphogenesis controlled by exogenous growth regulators were established. Agrobacterium rhizogenes strain LBA 9402 has been tested for its capacity to induce hairy roots of this monocotyledonae plant.     

Carotenogenic and abscisic acid biosynthesizing activity in a cell-free system

Abscisic acid is considered an apocarotenoid formed by cleavage of a C-40 precursor and subsequent oxidation of xanthoxin and abscisic aldehyde. Confirmation of this reaction sequence is still awaited, and might best be achieved using a cell-free system capable of both carotenoid and abscisic acid biosynthesis. An abscisic acid biosynthesizing cell-free system, prepared from flavedo of mature orange fruits, was used to demonstrate conversion of farnesyl pyrophosphate, geranylgeranyl pyrophosphate and all-trans-β-carotene into a range of β,β-xanthophylls, xanthoxin, xanthoxin acid, 1′,4′-trans-abscisic acid diol and abscisic acid. Identification of product carotenoids was achieved by high-performance liquid chromatography and on-line spectral analysis of individual components together with co-chromatography. Putative C-15 intermediates and product abscisic acid were identified by combined capillary gas chroma-tography-mass spectrometry. Kinetic studies revealed that β-carotene, formed from either famesyl pyrophosphate or geranylgeranyl pyrophosphate, reached a maximum within 30 min of initiation of the reaction. Thereafter, β-carotene levels declined exponentially. Catabolism of substrate β-carotene into xanthophylls, putative abscisic acid precursors and product abscisic acid was restricted to the all-trans-isomer. However, when a combination of all-trans- and 9-cis-β-carotene in the ratio 1:1 was used as substrate, formation of abscisic acid and related metabolites was enhanced. Biosyn-thetically prepared [¹⁴C]-all-trans-violaxanthin, [¹⁴C]-all-trans-neoxanthin and [¹⁴C]-9′-cis-neoxanthin were used as substrates to confirm the metabolic interrelationship between carotenoids and abscisic acid. The results are consistent with 9′-cis-neoxan-thin being the immediate carotenoid precursor to ABA, which is oxidatively cleaved to produce xanthoxin. Formation of abscisic aldehyde was not observed. Rather, xanthoxin appeared to be converted to abscisic acid via xanthoxin acid and 1′,4′-trans-abscisic acid diol. An alternative pathway for abscisic acid biosynthesis is therefore proposed.     

Physiology and Chemistry of Substances Accelerating Abscission in Senescent Petioles and Fruit Stalks

Senescent petioles of Coleus rehneltianus Berger. Phaseolus vulgaris L. cv. Saxa. Acer pseudoplatanus L., and senescent fruit stalks of Malus domestica Borkh. cv. Golden Delicious contain at least three abscission accelerating substances, which were isolated by extraction with methanol or with water and by diffusion into agar. They were purified by thin-layer chromatography and bioassayed in a special abscission test using Coleus explants. Two of these abscission accelerators could be conclusively identified by thin-layer chromatography and by gas chromatography as abscisic acid and xanthoxin. The third substance, which has acidic properties and is less polar than abscisic acid, could not be identified. The concentration and the absolute amount of abscisic acid in Coleus petioles were found to decrease during their development, young petioles having the highest concentration. No evidence was found that the three abscission accelerators or synthetic abscisic acid and xanthoxin affect the production of ethylene in Caleus explatns. The results obtained do not support the hypothesis that senescent petioles contain a specific “senescence factor”, which stimulates abscission via ethylene production.     

Formation and breakdown of ABA

The phytohormone, abscisic acid (ABA) is found in all photosynthetic organisms. The amount of ABA present is determined by the dynamic balance between biosynthesis and degradation: these two processes are influenced by development, environmental factors such as light and water stress, and other growth regulators. ABA is synthesized from a C₄₀ carotenoid precursor and the first enzyme committed specifically to ABA synthesis is a plastid- localized 9-cis-epoxycarotenoid dioxygenase, which cleaves an epoxycarotenoid precursor to form xanthoxin. Subsequently, xanthoxin is converted to ABA by two cytosolic enzymes via abscisic aldehyde, but there appears to be at least one minor alternative pathway. The major catabolic route leads to 8′-hydroxy ABA and phaseic acid formation, catalyzed by the cytochrome P450 enzyme ABA 8′-hydroxylase. In addition, there are alternate catabolic pathways via conjugation, 4′-reduction and 7′-hydroxylation. As a consequence of recent developments, the mechanism by which the concentration of hormonally active ABA is controlled at the cellular, tissue and whole plant level can now be analyzed in detail.     

Abscisic Aldehyde Is an Intermediate in the Enzymatic Conversion of Xanthoxin to Abscisic Acid in Phaseolus vulgaris L. Leaves

The enzymatic conversion of xanthoxin to abscisic acid by cell-free extracts of Phaseolus vulgaris L. leaves has been found to be a two-step reaction catalyzed by two different enzymes. Xanthoxin was first converted to abscisic aldehyde followed by conversion of the latter to abscisic acid. The enzyme activity catalyzing the synthesis of abscisic aldehyde from xanthoxin (xanthoxin oxidase) was present in cell-free leaf extracts from both wild type and the abscisic acid-deficient molybdopterin cofactor mutant, Az34 (nar2a) of Hordeum vulgare L. However, the enzyme activity catalyzing the synthesis of abscisic acid from abscisic aldehyde (abscisic aldehyde oxidase) was present only in extracts of the wild type and no activity could be detected in either turgid or water stressed leaf extracts of the Az34 mutant. Furthermore, the wilty tomato mutants, sitiens and flacca, which do not accumulate abscisic acid in response to water stress, have been shown to lack abscisic aldehyde oxidase activity. When this enzyme fraction was isolated from leaf extracts of P. vulgaris L. and added to extracts prepared from sitiens and flacca, xanthoxin was converted to abscisic acid. Abscisic aldehyde oxidase has been purified about 145-fold from P. vulgaris L. leaves. It exhibited optimum catalytic activity at pH 7.25 in potassium phosphate buffer.     

OsCESA9 conserved‐site mutation leads to largely enhanced plant lodging resistance and biomass enzymatic saccharification by reducing cellulose DP and crystallinity in rice

Genetic modification of plant cell walls has been posed to reduce lignocellulose recalcitrance for enhancing biomass saccharification. Since cellulose synthase (CESA) gene was first identified, several dozen CESA mutants have been reported, but almost all mutants exhibit the defective phenotypes in plant growth and development. In this study, the rice (Oryza sativa) Osfc16 mutant with substitutions (W481C, P482S) at P‐CR conserved site in CESA9 shows a slightly affected plant growth and higher biomass yield by 25%–41% compared with wild type (Nipponbare, a japonica variety). Chemical and ultrastructural analyses indicate that Osfc16 has a significantly reduced cellulose crystallinity (CrI) and thinner secondary cell walls compared with wild type. CESA co‐IP detection, together with implementations of a proteasome inhibitor (MG132) and two distinct cellulose inhibitors (Calcofluor, CGA), shows that CESA9 mutation could affect integrity of CESA4/7/9 complexes, which may lead to rapid CESA proteasome degradation for low‐DP cellulose biosynthesis. These may reduce cellulose CrI, which improves plant lodging resistance, a major and integrated agronomic trait on plant growth and grain production, and enhances biomass enzymatic saccharification by up to 2.3‐fold and ethanol productivity by 34%–42%. This study has for the first time reported a direct modification for the low‐DP cellulose production that has broad applications in biomass industries.     

Xanthoxin: A Growth Inhibitor in Light-grown Sunflower Seedlings, Helianthus annuus L.

Methanol extracts from light- and dark-grown sunflower seedlings,Helianthus annuus L., var. ‘Inra No. 6501’, weresubjected to solvent partition. The neutral diethyl ether fractionfrom the light-grown seedlings contained material(s) which inhibitedcress seed germination. Such inhibition was scarcely detectablein extracts of the etiolated seedlings. Sometimes inhibitionalso occurred in the petroleum spirit fraction. A mixture ofcis, trans- and trans, transxanthoxin showed the same partitioncharacteristics as the inhibiting substance(s); the latter co-chromatographedwith xanthoxin in all paper, thin layer, and high pressure liquidchromatography separation procedures tried. The inhibition wasalso detectable with the Avena colcoptile straight growth bioassay. It is concluded that xanthoxin is formed during illuminationof sunflower seedlings; its role in the phototropic responseof these seedlings is discussed.     

Production of a plasmin inhibitory substance by Scopolia japonica suspension cultures

A potent inhibitory agent against human plasmin, fibrinolytic proteinase, has been found in the extracts of callus tissue of Scopolia japonica. Effects of cultural conditions on cell growth and production of the plasmin inhibitory substance by this cell line in suspension cultures were examined in MurashigeSkoog's medium. More than l.5 mg of the inhibitor, as t-amino cyclohexane carboxylic acid, a synthetic plasmin inhibitor, were observed to accumulate per ml of medium containing 0.83 g of NH₄NO₃ and 7.6 g of KNO₃ per liter as well as suitable levels of growth hormones. Addiction of antibiotics and deformers were examined in preliminary tests for large scale cultivation. Semicontinuous culture on a small scale in a glass cylinder, was also tested and growth rate of 1.29 g/liter/day (by dry wt) was obtained. Plasmin inhibitory activities in the extracts of the results intact plant and in cultured cells of S. japonica were compared and the results indicated that cell suspension culture was superior to extraction the natural plant for inhibitor production.     

Characteristics of plant communities containing St. John’s wort (<Emphasis Type="Italic">Hypericum perforatum</Emphasis> L.) in the Saratov Region

Analysis of 28 plant communities containing Hypericum perforatum L. in five different biotope types has been performed in 17 districts of the Saratov Region. A total of 325 species from 197 genera of 52 vascular plant species have been recorded, with plants characteristic of forest-edge, steppe, and anthropogenic biotopes prevailing in most communities. It has been shown that H. perforatum at the southeastern boundary of its range behaves as an explerent species. The main factor limiting its growth in biotopes of a certain type is the degree of disturbance in the structure of corresponding communities rather than by their taxonomic composition.     

The short-chain alcohol dehydrogenase ABA2 catalyzes the conversion of xanthoxin to abscisic aldehyde

Mutants able to germinate and perform early growth in medium containing a high NaCl concentration were identified during the course of two independent screenings and named salt resistant (sre) and salobre?o (sa?). The sre and sa? mutants also were able to germinate in high-osmoticum medium, indicating that they are osmotolerant in a germination assay. Complementation analyses revealed that sre1-1, sre1-2, sa?3-1, and sa?3-2 were alleles of the abscisic acid (ABA) biosynthesis ABA2 gene. A map-based cloning strategy allowed the identification of the ABA2 gene and molecular characterization of four new aba2 alleles. The ABA2 gene product belongs to the family of short-chain dehydrogenases/reductases, which are known to be NAD- or NADP-dependent oxidoreductases. Recombinant ABA2 protein produced in Escherichia coli exhibits a K(m) value for xanthoxin of 19 micro M and catalyzes in a NAD-dependent manner the conversion of xanthoxin to abscisic aldehyde, as determined by HPLC-mass spectrometry. The ABA2 mRNA is expressed constitutively in all plant organs examined and is not upregulated in response to osmotic stress. The results of this work are discussed in the context of previous genetic and biochemical evidence regarding ABA biosynthesis, confirming the xanthoxin-->abscisic aldehyde-->ABA transition as the last steps of the major ABA biosynthetic pathway.     

Expression of a Chinese cabbage cysteine proteinase inhibitor, BrCYS1, retards seed germination and plant growth in transgenic Tobacco plant

Phytocystatins are plant cysteine proteinase inhibitors that regulate endogenous and heterologous cysteine proteinases of the papain family. A cDNA encoding the phytocystatin BrCYS1 (Brassica rapa cysteine proteinase inhibitor 1 ) has been isolated from Chinese cabbage (B. rapa subsp.pekinensis) flower buds. In order to explore the role of this inhibitory enzyme, tobacco plants (Nicotiana tabacum L. cv. Samson) containing altered amounts of phytocystatin were generated by over-expressingBrCYS1 cDNA in either the sense or the antisense configuration. The resulting plants hadin vitro enzyme inhibitory activities that were over 10% of those detected in wild type plants. The transgenic plants exhibited retarded seed germination and seedling growth and a reduced seed yield, whereas these properties were enhanced in antisense plants. These data suggest that BrCYS1 participates in the control of seed germination, post-germination and plant growth by regulating cysteine peptidase activity.     

NAD-Dependent and NADP-Dependent Alcohol Dehydrogenases in Escherichia coli

The isomeric ratio and level of natural xanthoxin (XAN) in tomato plants (Lycopersicon esculentum) were examined by a more reliable analytical method than has been reported before. Efforts were made to avoid artificial isomerization between c-XAN and t-XAN throughout the isolation, derivatization and GC-MS procedures. Natural XAN was separated from contaminating chlorophylls before rev. HPLC purification, derivatized to abscisic acid methyl ester (MeABA) in four chemical steps, and quantified with the deuterium-labeled internal standards on clear and reproducible full GC-EI-MS. It was revealed that the isomeric composition of natural XAN was exclusively shifted to c-XAN. The level of c-XAN was higher and more significantly induced by water stress in older plants. The significant role of c-XAN as an ABA biosynthetic precursor is suggested.