Phytochrome and hormonal control of expansion and greening of etiolated wheat leaves

Summary Unrolling of etiolated wheat leaf segments is stimulated by short periods of exposure to red light. Both gibberellic acid and kinetin will stimulate unrolling in the dark, whereas abscisic acid (ABA) inhibits the unrolling response to these two hormones and to red light. Exposure to 5 minutes of red light leads to a rapid increase in endogenous gibberellin levels in etiolated wheat leaves, and this increase is followed by a rapid decline. Pre-treatment with ABA inhibits the increase in gibberellin levels in response to red light, but the ihibitory effect of ABA on unrolling cannot be ascribed only to its effect on gibberellin levels. Pre-treatment with red light reduces the lag-phase in chlorophyll development when wheat leaf segments are subsequently exposed to white light; the effect of red light may be replaced by pre-treatment with kinetin, but gibberellic acid is relatively ineffective in this respect.     

Effects of Growth Regulators on Ribonucleic Acid Metabolism of Barley Leaf Segments

Illumination or gibberellic acid treatment of etiolated barley leaf segments stimulates unrolling and results in an increased level of RNA. In contrast, segments treated with abscisic acid do not unroll and have a lower content of RNA. Gibberellic acid treatment enhanced the capacity of segments to incorporate radioactivity from ³²P-orthophosphate into all the RNA components detected by gel electrophoresis; abscisic acid greatly restricted the incorporation of precursors into all the RNA fractions. In conjunction with a changed capacity for RNA synthesis it was observed that abscisic acid-treated segments had a lowered soluble DNA-dependent RNA polymerase level in comparison to gibberellic acid-treated or illuminated segments. However, the influence of growth regulators on RNA polymerase content of the segments was associated with general effects on protein level rather than a specific effect on the synthesis of polymerase enzyme.     

Barley leaf unrolling. The proline connection

Unrolling of 1 cm sections, taken between 3 and 4 cm from the apex, of 6-day-old, etiolated barley leaves, was promoted by blue (426 nm) and red (658 nm) light. Accompanying such unrolling was a reduction in the level of the free proline of the tissue. When leaf unrolling was prevented by irradiation with far-red (728 nm) light, or treatment with abscisic acid (ABA) following red light irradiation, the level of proline remained more or less unchanged, at the level of the untreated, dark controls. The proline analogue, azetidine carboxylic acid (AZC) powerfully inhibited the light induced leaf opening, emphasizing the significance of proline-containing, structural and functional proteins in barley leaf unrolling. The inhibition imposed by AZC is partially reversible by added proline.     

Polysomal changes in developing wheat leaves

Summary When leaf sections of 7-day old dark grown wheat leaves were incubated in white light, they unrolled and greened. Gibberellic acid was able to replace the light requirement and abscisic acid (ABA) inhibited the response to light. The percentage of ribosomes occurring as polysomes increased in response to light but not in response to GA₃ treatment. Although ABA inhibited the unrolling and greening in light, it did not cause a preferential decrease or inhibition of polysome formation.     

The effect of δ-aminolevulinic acid on red light-induced unrolling of dark-grown barley leaf sections

Unrolling of sections from dark grown barley leaves ( Hordeum vulgare L. cv. Klages) was stimulated by red irradiation. The unrolling started after a lag phase of 6-8 h and was completed after 24 h. The effect of several keto and amino acids on leaf unrolling was compared with their effect on coleoptile segment expansion growth. Of the substances tested δ-aminolevulinic acid had the most inhibitive effect on leaf unrolling and the least inhibitive effect on coleoptile segment expansion growth. Prolonged treatment caused a strong inhibition of the unrolling but with a short tretment, a stimulation of the unrolling occurred. The inhibitive effect of δ-aminolevulinic acid was only found if the treatment started within 6-8 h after the red irradiation. Kinetin and gibberellic acid could decrease the inhibitory effect of δ-aminolevulinic acid. A possible role for δ-aminolevulinic acid working as a triggering substance for leaf unrolling is discussed.     

The hormonal control of wheat leaf unrolling

Summary The unrolling of etiolated wheat leaf sections in the dark is stimulated by the application of gibberellic acid (GA₃). GA₃ is most effective if applied for a short time at the beginning of incubation. Kinetin also stimulated leaf unrolling in the dark. AMO1618 and CCC inhibit red light and kinetin-stimulated unrolling. Gibberellin-like substances extracted from red light-treated leaf tissue are effective in stimulating leaf unrolling.Ethylene production in leaf sections is stimulated by IAA, GA₃ and kinetin and inhibited by ABA. A brief exposure to red light decreases the ability of the tissue to produce ethylene. It is concluded that ethylene plays no important role in the control of leaf unrolling by red light or by the application of hormones.Holder of a Science Research Council Studentship.     

An increase in ethylene sensitivity is associated with jasmonate-promoted senescence of detached rice leaves

The role of ethylene in jasmonate-promoted senescence of detached rice leaves was investigated. Ethylene production in methyl jasmonate-treated leaf segments of rice was lower than in the control leaves. Treatment of leaf segments with silver nitrate or/and silver thiosulfate, inhibitors of ethylene action, inhibited methyl jasmonate-, jasmonic acid-, linolenic acid-, and abscisic acid-promoted senescence of detached leaves. We suggest that an increase in ethylene sensitivity, but not ethylene level, is the initial event triggering the enhanced senescence by jasmonates of detached rice leaves.Abbreviations JA jasmonic acid - MJ methyl jasmonate - STS silver thiosulfate - ABA abscisic acid     

Phytochrome-controlled Leaf Unrolling and Protein Synthesis

In the primary leaf sections of etiolated wheat (Triticum aestivum L.) seedlings, red light-induced unrolling is accompanied by an increase in incorporation of ¹⁴C-leucine into protein. By differential centrifugation, the unrolling response was found to be closely related to incorporation of the amino acid into the supernatant fraction (105,000g). Cycloheximide and chloramphenicol inhibit both leaf unrolling and synthesis of the supernatant protein, although chloramphenicol exerts its effect more strongly on the fraction which presumably contains the plastids. In a barley (Hordeum vulgare L.) albino mutant completely devoid of ribulose diphosphate carboxylase activity, only incorporation of ¹⁴C-leucine into the supernatant fraction is substantially promoted by red light. This mutant exhibits the photoresponse of leaf unrolling.     

Effects of growth regulators and light on chloroplasts NAD(P)H dehydrogenase activities of senescent barley leaves

The activities NADH and NADPH dehydrogenases were measured with ferricyanide as electron-acceptor (NADH-FeCN-ox and NADPH-FeCN-ox, respectively) in mitochondria-free chloroplasts of barley leaf segments after receiving various treatments affecting senescence. NADPH-FeCN-ox declined during senescence in the dark, in a way similar to chlorophyll and Hill reaction, and increased when leaf segments were incubated at light. These results suggest that NADPH-FeCN-ox is related to some photosynthetic electron transporter activity (probably ferredoxin-NADP⁺ oxidoreductase). In contrast, NADH-FeCN-ox is notably stable during senescence in the dark and at light. This activity increased during incubation with kinetin or methyl-jasmonate (Me-JA) but decreased when leaf segments were treated with abscisic acid (ABA). The effects of the inhibitors of protein synthesis cycloheximide and chloramphenicol suggest that the changes of NAD(P)H dehydrogenase activities may depend on protein synthesis in chloroplasts. In senescent leaf, chloroplast NADH dehydrogenase might be a way to dissipate NADH produced in the degradation of excess carbon which is released from the degradation of amino acids.Abbreviations ABA abscisic acid - DCPIP 2,6-dichlorophenol-indo-phenol - DOC deoxycholate - Me-JA methyl jasmonate - NADH-FeCN-ox NADH ferricyanide oxidoreductase - NADPH-FeCN-ox NADPH ferricyanide oxidoreductase     

Influence of phytohormones and other effectors on proton extrusion by isolated protoplasts from rape leaves

The roles of phytohormones and fusicoccin in H⁺ extrusion by isolated protoplasts from rape leaves ( Brassica napus L. cv. Belinda) were investigated and compared to results obtained with leaf segments of the same plants. Net H⁺ release by protoplasts, which was at least partly due to ATPase activity, was enhanced by 10 μ M indole-3-acetic acid and reduced by 20 μ M abscisic acid, whereas fusicoccin (10 μ M ), brassinosteroid (3 μ M ), kinetin (20 μ M ) and gibberellic acid (10 μ M ) had no effect. Hormone effects and H⁺ release were not detectable with leaf segments from the same plants. However, using field-grown plants, indole-3-acetic acid and especially fusicoccin stimulated the acidification of the external medium by leaf segments. Hormonecontrolled H⁺ release by leaf cells is interpreted as the first step in acid-triggered and turgor-regulated cell growth.     

The Effect of Phytohormones on Chlorophyll(ide), Protochlorophyll(ide) and Carotenoid Formation in Greening Dark Grown Wheat Leaves

The influence of phytohormones on chlorophyll and carotenoid formation during the greening of irradiated dark grown wheat leaves (Triticum aestivum L. cv. Starke II Weibull) was studied. Leaves were floated on solutions of abscisic acid, gibberellic acid and kinetin for 24 h. The chlorophyll and carotenoid contents were determined during a subsequent period of 48 h of continuous irradiation. Leaves treated with abscisic acid showed a longer lag phase and a lower rate of accumulation of chlorophyll as compared to the control than did leaves treated with gibberellic acid and kinetin. The carotenoid content was low both in leaves treated with abscisic acid and in those treated with gibberellic acid. Treatment with abscisic acid lowered the protochlorophyllide regeneration after a saturating light flash while gibberellic acid as well as kinetin had no effect. The influence of ABA was partly dependent on an increase of the wounded part of the cut leaf segments. The accumulation of protochlorophyllide in leaves treated with δ-aminolevulinic acid was not affected by the different hormonal treatments. These results suggest that the main effect of abscisic acid is probably outside the chloroplast, i.e. on the formation or transport of δ-aminolevulinic acid.     

Phytochrome-mediated swelling of etiolated leaf protoplasts and its possible biological significance

Summary Red light, mediated by the photoreceptor phytochrome, induces maize leaf unrolling as well as leaf expansion. Protoplasts prepared from maize leaves still in the rolled condition swell in a red-far red photoreversible manner indicating that phytochrome mediates this phenomenon. To determine if protoplast swelling is related to leaf unrolling, leaf expansion, or both, we compared red-light induced swelling of protoplasts from rolled maize leaves to protoplasts prepared from tissues that are known to grow in response to light but do not unroll. We also compared the swelling response of protoplasts from rolled vs. unrolled leaves. In all cases, we found that swelling correlated with the unrolling response and not leaf expansion.     

The effects of abscisic acid,kinetin and 5-fluorouracil on ribonucleic acid and protein synthesis in senescing radish leaf disks

Summary The effects of abscisic acid and kinetin on RNA synthesis in senescing radish leaf disks were investigated using the improved resolution afforded by polyacrylamide gel electrophoresis. Kinetin stimulated and abscisic acid inhibited incorporation of radioactivity into cytoplasmic ribosomal RNA and soluble RNA. Chloroplast ribosomal RNA synthesis appeared to be confined to the period of leaf expansion and was not detected in fully mature leaves. The effects of kinetin in retarding and of abscisic acid in accelerating leaf senescence were not altered by the inhibition of cytoplasmic ribosomal RNA synthesis with 5-fluorouracil. Following inhibition of cytoplasmic ribosomal RNA synthesis with 5-fluorouracil, kinetin stimulated and abscisic acid inhibited incorporation of radioactivity into polydisperse RNA. These results are discussed in relation to the possible mode of action of kinetin and abscisic acid in senescing leaf tissue.     

Abscisic Acid and stomatal regulation

The closure of stomata by abscisic acid was examined in several species of plants through measurements of CO₂ and H₂O exchange by the leaf. The onset of closure was very rapid, beginning at 3 minutes from the time of abscisic acid application to the cut base of the leaf of corn, or at 8 or 9 minutes for bean, Rumex and sugarbeet; rose leaves were relatively slow at 32 minutes. The timing and the concentration of abscisic acid needed to cause closure were related to the amounts of endogenous abscisic acid in the leaf. Closure was obtained in bean leaves with 8.9 picomoles/cm². (+)-Abscisic acid had approximately twice the activity of the racemic material. The methyl ester of abscisic acid was inactive, and trans-abscisic acid was likewise inactive. The effects of stress on levels of endogenous abscisic acid, and the ability of very small amounts of abscisic acid to cause rapid closure suggests that stomatal control is a regulatory function of this hormone.     

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.     

The role of abscisic Acid in cross-adaptation of tobacco plants

Tobacco plants (Nicotiana rustica L.) pre-exposed to leaf dehydration, mineral deprivation, salination, or BO₃³⁻ toxicity exhibited increased resistance to subzero temperature and to reduced oxygen in the root medium. The stressed plants all showed an elevated content of leaf abscisic acid. Upon transfer of mineral deprived and salinated plants to prestress conditions, a decline in leaf abscisic acid content to prestress levels took place together with a loss of the increased resistance to subzero temperature and to deprivation of root oxygen. Treatment with abscisic acid by direct application to the leaves or by addition to the root medium improved leaf resistance to subzero temperature and to deprivation of root oxygen. A common hormone-regulation mechanism involving abscisic acid is suggested for this phenomenon of “cross-adaptation” by which a given stress confers increased resistance to other, apparently unrelated stresses.     

Stomatal response to drying soil in relation to changes in the xylem sap composition of Helianthus annuus. II. Stomatal sensitivity to abscisic acid imported from the xylem sap

Sunflower plants [Helianthus annuus L.) were subjected to soil drought. Leaf conductance declined with soil water content even when the shoot was kept turgid throughout the drying period. The concentration of abscisic acid in the xylem sap increased with decreasing soil water content. No general relation could be established between abscisic acid concentration in the xylem sap and leaf conductance due to marked differences in the sensitivity of leaf conductance of individual plants to abscisic acid from the xylem sap. The combination of these results with data from Gollan, Schurr & Schulze (1992, see pp. 551–559, this issue) reveals close connection of the effectiveness of abscisic acid as a root to shoot signal to the nutritional status of the plant.     

Effect of vanadate on bean leaf movement, stomatal conductance, barley leaf unrolling, respiration, and phosphatase activity

Vanadate (Na₃ VO₄) inhibits leaf movement and stomatal conductance of Phaseolus vulgaris L. cv. Carlos Favorit in light-dark cycles as well as photomorphogenetic leaf unrolling of Hordeum vulgare L. cv. Rupal. Inhibition was 50% by 10 to 100 micromolar vanadate and 100% by millimolar vanadate. Leaf unrolling was also inhibited by oligomycin and diethylstilbestrol.