Control of Senescence in Rumex Leaf Discs by Gibberellic Acid

Hormones which inhibit senescence of Rumex leaf tissue in the dark include gibberellin A3, and the cytokinins 6-benzylamino purine and zeatin. These hormones inhibit respiratory metabolism in this tissue, but do not change the pattern or total amount of oxygen consumption during senescence. Abscisic acid, a senescence accelerator, correspondingly stimulates oxygen consumption. This correlation of senescence rate and respiration rate holds with regard to the hormone concentrations effective and the continued activity of the hormones when added after the lag phase of chlorophyll breakdown. Transfer experiments show that the respiratory inhibition due to gibberellin A3 and the promotion due to abscisic acid become established within 3 hours of hormone addition. When gibberellin A3 and zeatin were rapidly added to narrow strips of tissue, no inhibitions of oxygen uptake were observed in the first 12 minutes. Senescence-inhibiting concentrations of sucrose strongly stimulate respiratory meabolism, raise the respiratory quotient, and cause inhibition of chlorophyll and protein breakdown which is distinct from the effect of gibberellins or cytokinins.It is proposed that the gibberellins and cytokinins may act by inhibiting a rate-limiting process in the metabolism of starving leaf tissue. This rate-limitation hypothesis envisions a possibly subtle action of the hormone which retards but does not alter the pattern of macromolecular changes during cell autolysis.The earlier literature established that respiration is active during the senescence of attached and detached leaves (24). Oxygen consumption can show a peak during yellowing which is superficially similar to the climacteric maximum observed in fruits. Senescence of detached grass leaves (Pennisetum sp., Andropagon sp., and Avena) correlates with and is in some way dependent on oxidative metabolism, since atmospheres of N2 or CO2 strongly inhibit Chl and protein breakdown (13,23 MATERIALS AND METHODSRespirometry. Expanded leaves were excised from Rumex crispus or R. obtusifolius plants grown from seed in a greenhouse as previously described (6). In the sucrose experiments and in later experiments, leaves were surface-sterilized by soaking 5 min in distilled water, 1 to 4 min in a 0.5% w/v sodium hypochlorite commercial bleach solution, and rinsed twice in sterile distilled water. Samples of 10 or 20 discs of leaf tissue 6.25 mm in diameter were excised from the leaves with a paper punch and floated on test solutions in sterile plastic Petri dishes or in sterilized respirometer vessels. Solutions were usually filter-sterilized and were replaced before respirometry to avoid accumulation of bacteria which can contribute significantly to the oxygen consumption, especially during the later stages of tissue senescence. Tissue samples were incubated at 30 C in darkness or under a dim incandescent bulb or green safelight for brief intervals while reading the manometers.Oxygen consumption at 30 C was measured by the direct method in a Warburg-Barcroft constant vol...

Section: Cambridge Massachusetts 02138mentioning

confidence: 99%

Energy Metabolism of Rumex Leaf Tissue in the Presence of Senescence-regulating Hormones and Sucrose

Goldthwaite¹

1974

“…The chlorophyll retention assay employed by Chua (4) suffers from a lack of specificity (11). Both gibberellins (6,17) and soluble peptones (17) are more active than cytokinins in retarding the senescence of Rumex leaves, and EDTA retards chlorophyll loss in darkmaintained barley leaves (9). This appears to be another case in which the chlorophyll retention assay is too unspecific to be used in evaluating proposed cytokinins.…”

Section: Resultsmentioning

confidence: 84%

Lack of Cytokinin Activity of Quinolinol Sulfate

Kraft

Ellis

1971

Both purine (1, 10) and urea (3) derivatives have been shown by various bioassays to be active cytokinins, the latter leading Bruce and Zwar (3) to hypothesize -NH--CO-NH-R as the active portion of the cytokinin molecule. Currently, cytokinins which have been extracted from plant materials, particularly from transfer RNA, are all N6-substituted purines (7,14). Fox and Chen, after demonstrating that benzyladenine, an N6-substituted purine, is incorporated into transfer RNA, hypothesized that cytokinins structurally complete certain inactive transfer RNA molecules, allowing them to function normally (5). While there is disagreement about this mechanism (8), the importance of purines is undisputed.Chua (4), using the chlorophyll retention assay, has recently identified 8-quinolinol sulfate (Fig. 1), an antimicrobial agent, as a cytokinin with maximal activity at 0.1 to 1 mm. This chemical differs markedly in structure from the usual purine nucleus, and obviously could not function through incorporation into tRNA. Chua suggests, somewhat unclearly, that the controlling activity of 8-quinolinol sulfate may be as a chelator, interfering in specific protein-metal cofactor relationships. In contrast, Rameshwar and Steponkus (16) report that 8-quinolinol sulfate cannot replace kinetin in stimulating the growth of soybean callus.Since the establishment of 8-quinolinol sulfate as a true cytokinin would throw considerable doubt on current ideas of the mechanism of cytokinin action, a more thorough evaluation of this compound, including possible interactions with known cytokinins, is desirable. In view of the fact that the conflicting results mentioned previously are based on only one bioassay performed in each laboratory, we decided to use several assays, each of which is highly specific (11, 18), and conducted in axenic conditions: the growth of tobacco pith (15) and soybean callus (13), and the production of buds by moss protonema (2). METHODS AND MATERIALSThe tobacco callus bioassay was carried out on the media of Murashige and Skoog (15), with the addition of 2 mg IAA/ liter and 1.6% agar. Pith was excised, by using a cork borer, from randomized segments of ornamental tobacco stem (Nicotiana tabacum L. cv. Assimis, obtained from Pennsylvania State University), cut into 0.5 x 0.3 cm pieces and 1 piece placed on approximately 35 ml of medium in a Petri dish. Final weights were taken after 4 to 5 weeks of growth in the dark.Soybean callus (Glycine max (L.) Merr., var. Acme) was obtained from Dr. H. Kende, AEC Plant Research Laboratory at Michigan State University. Pieces of callus, approximately 1.0 x 0.5 cm, were placed on 50 ml of solidified media (13) in 125-ml Erlenmeyer flasks and grown under continuous light for 6 to 7 weeks, when final weights were taken.The assay employing moss protonema was carried out as described by Brandes and Kende (2) on the media of Wood et al. (20). Spores of Funaria hygrometrica (L.) Sibth, also supplied by Dr. H. Kende, were grown in Petri plates for 26 days on the basal medium, and then ...

“…He concludes that cytokinins, but not GA3, interact with ABA in this system. GA, and cytokinins retard senescence, as measured by Chi decline, in Ruinex, Taraxacum, and Tropaeolum leaf discs (2,8). ABA has been shown to accelerate Chl breakdown in leaf discs from these and 18 other species (2,3,5).…”

Section: Introductionmentioning

confidence: 99%

“…Since Chl breakdown kinetics in Rumex (8) and other species is not homogeneous, we have reinvestigated the question of interaction of cytokinins and GA3 with ABA-utilizing rate measurements (13) and using zeatin instead of kinetin as our cytokinin. Chl breakdown is taken as a valid measure of the rate of tissue senescence in this study because it correlates well with proteolysis and respiratory changes in Rumnex leaf discs senescing rapidly on H20 or ABA or slowly on GA3 or a cytokinin (3,7).…”

mentioning

confidence: 99%

See 1 more Smart Citation

A Kinetic Analysis of the Effects of Gibberellic Acid, Zeatin, and Abscisic Acid on Leaf Tissue Senescence in Rumex

Manos

Goldthwaite²

1975

Hormones which inhibit senescence in Rumex leaf tissue in the dark include gibberellic acid and the cytokinin zeatin. Abscisic acid accelerates senescence in this tissue. Other workers have proposed that cytokinins, but not gibberellins, interact with abscisic acid in senescing Rumex leaf tissue. The present study reinvestigates the question of interaction using measurements of chlorophyll degradation kinetics as parameters of senescence rate and draws the conclusion that neither zeatin nor gibberellic acid interact with abscisic acid in this system. In support of this conclusion are these results. Zeatin clearly cannot overcome the effects of abscisic acid when hormone solutions are replaced every other day. The kinetics of chlorophyll breakdown for tissue treated with unreplaced saturating zeatin solutions is different from that of tissue exposed to saturating zeatin plus abscisic acid. The observed rates of chlorophyll breakdown for tissue treated with abscisic acid and zeatin agree closely with predicted rates using a multiplicative model for independent action of the two hormones.Zeatin solutions, when replaced every other day, show up to a 550-fold increase in effective concentration in the retardation of senescence. Less than a 10-fold increase could be accounted for by the addition of more zeatin molecules to the tissue. A nonbiological inactivation of zeatin or the production of an inhibitor of zeatin action by the tissue could not be demonstrated. It seems that zeatin is metabolically inactivated or sequestered in this tissue. Two chemical substances are reported to interact if the response to both of them applied simultaneously is not equal to the sum of their separately evoked responses. The sum of the responses for two independent variables is not necessarily additive, depending rather on the system under study, and it may be multiplicative or more complex. Since plant responses seldom have mechanistic models worked out for their action, tests of negative interactions or antagonism are usually simplified and aimed at showing that at a saturating concentration of one substance, the effect of a second is nullified. This result is taken to demonstrate an interaction (12).The most desirable response to measure in such studies is the rate of response, because this avoids misinterpretations due to nonlinear kinetics and to lag or latent periods in the system. It is also theoretically the best response because the rate should be most sensitive to changes in effective hormone concentration. Nevertheless, some interesting studies have been made in which rates were not directly measured. Instead, after an incubation of a tissue on the growth regulator being tested, a single time point measurement is made of the response. For example, Khan (10) has used this approach to show that kinetin and benzyladenine, at sufficiently high concentrations, can overcome the inhibition of a-amylase activity accumulated after 4 days in barley seeds caused by ABA. He concludes that cytokinins, but not GA3, interact wit...