George G. Laties

Quantification of hydrogen peroxide in plant extracts by the chemiluminescence reaction with luminol

Abstract The chemiluminescence of luminol (3-aminophthalhydrazide) with H 2 O 2 has been used to quantify endogenous amounts of H 2 O 2 in plant tissues. The reaction is linear over at least three orders of magnitude between 10 −5 and 10 −2 M H 2 O 2 . Interference by coloured compounds in the crude extract is calibrated by a purification step with Dowex AG 1-X8. The extract is calibrated with an internal H 2 O 2 standard, and the specificity verified by H 2 O 2 purging with catalase. The minimum delectability for H 2 O 2 of this assay is at least 1 ng, corresponding to 0.1–1 g fresh material. Data are presented for the levels of H 2 O 2 in potatoes after treatment with oxygen and ethylene, in tomatoes before and after ripening and in untreated germinating castor beans as well as in beans treated with aminotriazol to inhibit catalase activity. Though data using the titanium test are generally confirmed, the method presented here has the advantage of higher sensitivity and specificity.

Cellulase gene expression in ripening avocado fruit: the accumulation of cellulase mRNA and protein as demonstrated by cDNA hybridization and immunodetection

SummaryA cDNA library was constructed from poly(A)+RNA of ripe avocado fruit. Colony hybridization identified a number of ripening specific clones of which one, pAV5, was shown to be specific for cellulase. Hybrid selection with pAV5 provided a message from ripe fruit that on in vitro translation yielded a polypeptide of 53kD, comigrating with purified avocado cellulase on SDS polyacrylamide gel electrophoresis. The translation product was selectively immunoprecipitated by antiserum to purified avocado cellulase. Immunoblotting of unripe and ripe avocado fruit extracts following SDS-PAGE showed a plentiful immunoreactive polypeptide in ripe fruit, and essentially none in unripe fruit. Hybridization of pAV5 to poly(A)+-RNA from unripe and ripe avocado fruit demonstrated that there is at least a 50-fold increase in the cellulase message concentration during ripening. Thus, the expression of cellulase enzyme activity during ripening is regulated by the appearance of mRNA coding for cellulase rather than by either translational or post-translational control mechanisms.

Gene expression during fruit ripening in avocado.

The poly(A) +RNA populations from avocado fruit (Persea americana Mill cv. Hass) at four stages of ripening were isolated by two cycles of oligo-dT-cellulose chromatography and examined by invitro translation, using the rabbit reticulocyte lysate system, followed by two-dimensional gel electrophoresis (isoelectric focusing followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis) of the resulting translation products. Three mRNAs increased dramatically with the climacteric rise in respiration and ethylene production. The molecular weights of the corresponding translation products from the ripening-related mRNAs are 80,000, 36,000, and 16,500. These results indicate that ripening may be linked to the expression of specific genes.

Nature and Control of Respiratory Pathways in Plants: The Interaction of Cyanide-Resistant Respiration with the Cyanide-Sensitive Pathway

Publisher Summary This chapter discusses the interaction of cyanide-resistant respiration with the cyanide-sensitive pathway. During conventional respiration, reducing equivalents from tricarboxylic acid cycle (TCA) intermediates in the matrix are transferred to the cytochrome chain via the internal, respiratory-linked NADH or succinic dehydrogenases. Electron passage through the cytochrome chain is sensitive to antimycin A and cyanide and is coupled to two or three phosphorylation sites. The respiratory chain is imbedded in the inner membrane of the mitochondrion, and during electron transport protons are translocated out of the matrix to create a proton motive force across the membrane, which drives phosphorylation. In addition to the cytochrome chain, the mitochondria of many higher, plant tissues, and fungi possess an alternative, cyanide-resistant electron transport pathway. This pathway is also insensitive to antimycin A, suggesting that it branches from the main respiratory chain on the substrate side of the b cytochromes.

Induction by ethylene of cyanide-resistant respiration

Ethylene and cyanide induce an increase in respiration in a variety of plant tissues, whereas ethylene has no effect on tissues whose respiration is strongly inhibited by cyanide. It is suggested that the existence of a cyanide-insensitive electron transport path is a prerequisite for stimulation of respiration by ethylene.

α-Oxidation of endogenous fatty acids in fresh potato slices

Abstract The respiration of fresh potato slices is inhibited up to 30% by imidazole, an inhibitor of α-oxidation of long chain fatty acids; it is stimulated approximately threefold by the uncoupler, carbonylcyanide m -Cl-phenylhydrazone ( m -Cl-CCP). The stimulation by m -Cl-CCP is totally inhibited by imidazole. The release of 14 CO 2 from car☐yl-labelled long chain fatty acids is also stimulated by m -Cl-CCP in fresh slices, and inhibited by imidazole. The δ 13 C value of fresh tissue respiratory CO 2 —indicative of the 13 C/ 12 C ratio of the endogenous respiratory substrate—indicates lipid to be the predominant substrate in fresh slices, as well as in fresh slices treated with m -Cl-CCP. Aged slices are considerably less imidazole sensitive in all the above respects. The substrate for aged slice respiration as indicated by δ 13 C values is carbohydrate. The basal respiration in fresh potato slices apparently involves considerable lipid oxidation of a mixed nature, while the respiratory increment in the presence of m -Cl-CCP represents predominantly α-oxidation of long chain fatty acids.

Site-specific binding of a nuclear factor to the carrot extensin gene is influenced by both ethylene and wounding

Experiments conducted in vitro using the electrophoretic mobility shift assay have shown that a single region of the extensin gene of carrot (Daucus carota L.) interacts with a protein factor designated Extensin Gene Binding Factor-1 (EGBF-1) present in nuclear extracts obtained from carrot roots. This interaction is sequence-specific as judged by the failure of other plant gene sequences to compete with the extensin gene for EGBF-1 binding. The EGBF-1 activity is organspecific, not being expressed in nuclear extracts obtained from carrot leaves or stems. Both ethylene treatment and wounding of roots are shown to have a controlling influence on the expression of EGBF-1 activity in nuclear extracts. These results demonstrate that at least three distinct signals: ethylene treatment, wounding, and development, are important in determining the activity of EGBF-1 in nuclear extracts, and indicate a role for EGBF-1 in stress-related signal transduction and the regulation of extensin-gene expression.

The regulatory function of potato pyruvate dehydrogenase

Abstract The effect of selected metabolites on the activity of potato pyruvate dehydrogenase has been studied. NADH is a potent competitive inhibitor with respect to NAD, and the content of NAD and NADH in whole tissue suggests that the balance between them may afford a fine regulatory mechanism for pyruvate dehydrogenase. Acetalhyde is not inhibitory. While glyoxylate competitively inhibits pyruvate dehydrogenase, the inhibitory characteristics fail to make glyoxylate an effective regulator of pyruvate oxidation. Acetyl CoA is a moderate competitive inhibitor with CoA, and its effect is enhanced by NADH, ATP, ADP, and AMP inhibit pyruvate dehydrogenase by binding Mg 2+ .

Absorption and long distance transport by isolated stele of maize roots in relation to the dual mechanisms of ion absorption

SummaryIon absorption and transport by intact roots, isolated cortex and isolated stele were compared shortly after tissue isolation and after aging. Absorption isotherms in the low and in the high concentration range show that in stripped-stele, which absorbs at a very low rate immediately after isolation, the capacity of system 1 but not system 2 is built up with aging. In agreement with this result analysis of individual fluxes across plasmamembrane and tonoplast reveals that only the influx from the medium into the cytoplasm increases considerably with aging of stele. Changes observed in aging excised roots and in isolated cortex are much less significant. In spite of the increase of absorption with aging by isolated stele, long distance transport, which is essentially passive through freshly stripped stele, decreases with aging. The reported results reflect the marked permeability of the plasmamembrane of fresh isolated stele, and demonstrate the importance of the cortex as a tissue “collecting” ions for long distance transport. New evidence for the theory of symplasmatic transport of ions into the xylem vessels is thus provided.ZusammenfassungDie Ionenaufnahme durch intakte Wurzeln, isolierte Rinde und isolierte Zentralzylinder und der Ferntransport durch intakte und entrind ete Wurzeln wurden verglichen, und zwar kurz nach der Isolierung und nach Altern der Gewebe. Frisch isolierte Zentralzylinder akkumulieren Io nen nur in ganz geringem Maße oder überhaupt nicht. Von den beiden Systemen der metabolischen Ionenaufnahme, die in einem niedrigen (System 1: bis 0,5 meq/l) und in einem hohen Konzentrationsbereich (System 2: 1-50 meq/l) die Geschwindigkeit der Ionenaufnahme durch intakte Wurzeln und isolierte Wurzelrinde bestimmen, entwickelt sich während des Alterungsprozesses in isolierten Zentralzylindern System 1, nicht aber System 2.In Übereinstimmung mit diesem Befund zeigt die Analyse der Einzelfluxe am Plasmalemma und am Tonoplasten, daß nur der Influx aus der Außenlösung in das Cytoplasma beim Altern der Zentralzylinder beträchtlich ansteigt. Veränderungen beim Altern von abgeschnittenen, intakten Wurzeln und isolierter Rinde sind viel weniger ausgeprägt.Obwohl die Ionenaufnahme beim Altern isolierter Zentralzylinder steigt, verringert sich der Ferntransport, der bei frisch isolierten Zentralzylindern rein passiv ist. Die mitgeteilten Ergebnisse zeigen die ausgeprägte Permeabilität frisch isolierter Zentralzylinder und demonstrieren die Bedeutung der Wurzelrinde als ein Gewebe, das Ionen für den Ferntransport “sammelt”. Auf diese Weise werden neue Anhaltspunkte für die Theorie des symplasmatischen Transportes der Ionen in die Gefäße gewonnen.

The nature of the respiratory rise in slices of chicory root

Abstract 1. 1. The respiratory increment which arises during the incubation of thin pieces of chicory root has been shown to be qualitatively distinct from the initial respiration. Whereas the initial respiration is almost entirely malonate resistant, the developed respiration is completely malonate sensitive. 2. 2. The respiration of fresh tissue may be increased several fold by the uncoupling agent 2,4-dinitrophenol (DNP), and the rise so induced is malonate sensitive. The respiration rate of incubated tissue is unaffected by DNP. 3. 3. The malonate-sensitive increment arising on aging, and the malonate-sensitive increment induced in fresh tissue by DNP have been shown to be different. 4. 4. Malonate-treated aged tissue readily oxidizes malate, thereby restoring the uninhibited respiration rate. By contrast, malate fails to affect malonate-inhibited, DNP-treated fresh tissue. Mitochondrial preparations from both types of tissue oxidize malate equally. Evidence has been presented that fresh tissue is permeable to malate. 5. 5. The nucleotide composition of fresh and incubated tissue was examined. Adenosine triphosphate (ATP) proved the predominant nucleotide in both cases, and no significant difference in nucleotide prevalence or composition was noted. 6. 6. The suggestion has been made that the respiratory rise attending aging is initiated by the removal of a restraint on the phosphorylative activity of the tissue.