The Activity of α-Amylase in the Shoot and its Relation to Gibberellin-induced Elongation

The activity of α-analyses in various plant organs was examined and the relation- ship between the enzyme activity and the leaf sheath elongation of dwarf mutants of maize was investigated. It has been shown that α-amylase exists in various plant organs. Especially high activity was detected in the bean hypocotyl. The regional activity of a-amylase in the epicotyl of the pea and the hypocotyl of the morning glory was examined. Higher activity was observed in the regions closer to the cotyledons. In the first leaf sheath of d₅ mutants of maize, GA₃-treatment resulted in the promotion of α-amylase activity, and there was a parallelism between GA₃-induced elongation and α-amylase activity. Removal of the endosperm from seedlings did not influence the GA₃-indnced elongation of the leaf sheath or the promotion of α-amylase activity. From these results it is concluded that at least some of the α-amylase is actually formed in the leaf sheath, and that there exists a distinct parallelism between the GA₃-induced promotion of enzyme activity and leaf sheath elongation.     

Promotion of leaf sheath growth by gibberellic acid in a dwarf mutant of rice

The mechanism of gibberellin (GA)-induced leaf sheath growth was examined using a dwarf mutant of rice (Oryza sativa L. cv. Tan-ginbozu) treated in advance with an inhibitor of GA biosynthesis. Gibberellic acid (GA₃) enhanced the growth of the second leaf sheath, but auxins did not. Measurement of the mitotic index and cell size revealed that cell elongation rather than cell division is promoted by GA₃. Gibberellic acid increased the extensibility of cell walls in the elongation zone of the leaf sheath. It also increased the total amount of osmotic solutes including sugars in the leaf sheath, but did not increase the osmotic concentration of the cell sap, due to an accompanying increase in cell volume by water absorption. In the later stage of GA₃-induced growth, starch granules completely disappeared from leaf sheath cells, whereas dense granules remained in control plants. These findings indicate that GA enhances cell elongation by increasing wall extensibility, osmotic concentration being kept unchanged by starch degradation. Received: 28 August 1997 / Accepted: 16 October 1997     

Hormonal Response and Seed Viabil ity of Parteresia coarctata,a Brackish Water Species

Stimulation of α-amylase activity was observed in Porteresia coarctata immature seeds (20-day-old) when de-embryonated prewashed half seeds were incubated in media containing gibberellic acid (GA₃, 10^?5M). No such activity was observed in mature seeds even when GA₃ concentration was increased up to five fold. ABA suppressed the GA₃ enhanced α-amylase synthesis up to nearly 70% in the immature seeds. Absence of this enzyme activity in mature seeds may be due to high levels of ABA. The immature aleurone showed a 23 kD polypeptide induced by ABA.     

Effects of light, temperature, gibberellin (GA3) and their interaction on coleoptile and leaf elongation of tall, semi-dwarf and dwarf wheat

Near-isogenic wheat lines differing in height-reducing (Rht) alleles, in each of two cultivars, were used to investigate the effects of light intensity and of their interaction with temperature and GA₃ application, on the elongation of the coleoptile and the first seedling leaf. Darkness caused a conspicuous increase in the lengths of the coleoptile and of the sheath and lamina of the first leaf, in GA₃ treated and untreated seedlings of all genotypes grown at 11 and 25°C. The genotype effects and the effects of light intensity and GA₃ application on leaf length were ascribed entirely to their effects on the rate of leaf elongation since the duration of leaf elongation was not affected by these factors. Temperature elevation from 11 to 25°C caused a 55% shortening of the duration of leaf elongation and a concomitant increase in elongation rate, which diminished with increased genotypic dwarfness. Accordingly, temperature elevation resulted in a significant reduction in leaf-length of the light-grown dwarf genotypes and the dark-grown dwarf and semi-dwarf genotypes. It is suggested that this temperature × light × genotype interaction effect is due to environmental dependent upper limits of elongation rate set by the Rht alleles.Abbreviations PAR Photosynthetic Active Radiation     

Enzyme formation and release by isolated barley aleurone layers

Aleurone layers, with testa attached, were prepared from degermed, decorticated barley with the aid of a fungal enzyme preparation. The preparations appeared intact under the scanning electron microscope. By using antibiotics only in an early stage preparations were obtained uncontaminated by micro-organisms and which, when incubated under optimal conditions with gibberellic acid, GA₃, produced near-maximal amounts of α-amylase. The enzyme accumulated in the tissue before it was released into the incubation medium. Daily replacement of the incubation medium, containing GA₃, depressed the quantity of α-amylase produced. α-Amylase was also produced in response to gibberellins GA₁, GA₄ and GA₇ and, to a much lesser extent, helminthosporol and helminthosporic acid. A range of other substances, reported elsewhere to induce α-amylase formation, failed to do so in these trials. At some concentrations, glutamine marginally enhanced the quantity of enzyme formed during prolonged incubations. It is confirmed that α-glucosidase occurs in the aleurone layer and embryo of ungerminated barley, and increases in amount during germination. GA₃ is shown to enhance this increase. When embryos arc burnt, to prevent gibberellin formation, no rise in α-glucosidase levels occurs unless GA₃ is supplied to the grains. As the activity of α-glucosidase and other enzymes have been determined as ‘α-amylase’ by some assay methods, their alterations in activity in response to GA₃ necessitates a re-evaluation of the evidence for de novo) synthesis of α-amylase in aleurone tissue.     

Gibberellic acid (GA3)-induced enhancement of α-amylase activity in the aleurone of Shrunken-2 maize kernels

The aleurone of RB-3 shrunken-2 (sh2) maize kernels is deficient in α-amylase activity during germination, but exogenous applications of gibberellic acid (GA₃) (0.001–10 μm ) induced low levels of activity. The highest activity was measured in the aleurone of kernels treated with 10 μm GA₃ (14,600 ± 945 units), but was lower than untreated Starchy (Su) aleurone tissues (35,280 ± 5,010 units). On isoelectric focusing gels, no α-amylase isozymes were detected in the untreated sh2 aleurone using starch zymograms or immunoblots, but the 1.0 and 10 μm mm GA₃ treatments induced nearly all the isozymes (eight to ten) present in the Su aleurone. There was a very low level of α-amylase mRNA in the untreated sh2 aleurone, an intermediate level in the 1.0 μm GA₃-treated sh2 aleurone, and the highest level in the untreated Su aleurone. On the confocal microscope, the 1.0 μm GA₃-treated aleurone cells had enhanced levels of cytoplasmic membranes and RNA compared to untreated sh2 aleurone cells. The 1.0 μm GA₃ treatment also induced shoot elongation in the sh2 seedlings. The data demonstrate that the sh2 aleurone is deficient in its function to produce α-amylases, and exogenous GA₃ can partially restore cell function in the sh2 kernels.     

Identification and characterization of a gibberellin-regulated protein,which is ASR5, in the basal region of rice leaf sheaths

Gibberellins (GAs) regulate growth and development in higher plants. To identify GA-regulated proteins during rice leaf sheath elongation, a proteomic approach was used. Proteins from the basal region of leaf sheath in rice seedling treated with GA₃ were analyzed by fluorescence two-dimensional difference gel electrophoresis. The levels of abscisic acid-stress-ripening-inducible 5 protein (ASR5), elongation factor-1 beta, translationally controlled tumor protein, fructose-bisphosphate aldolase and a novel protein increased; whereas the level of RuBisCO subunit binding-protein decreased by GA₃ treatment. ASR5 out of these six proteins was significantly regulated by GA₃ at the protein level but not at the mRNA level in the basal region of leaf sheaths. Since this protein is regulated not only by abscisic acid but also by GA₃, these results indicate that ASR5 might be involved in plant growth in addition to stress in the basal regions of leaf sheaths.     

Interaction between Biological Activities on Rice Seedlings of Dehydroabietic Acid Derivatives and Gibberellin A3

Effects of hydrofluorene and hydrophenanthrene compounds derived from dehydroabietic acid on the second leaf sheath growth of rice seedlings were examined in the presence and absence of gibberellin A₃(GA₃). In the absence of GA₃, nineteen compounds at 100 ppm inhibited more than 20% the growth of normal rice seedlings. In the presence of GA₃ (1.5 ppm) with dwarf rice seedlings, nine compounds at 500 ppm suppressed the elongation caused by the hormone, and a compound was slightly promotive. Then, three compounds were selected and subjected to the bioassay under various conditions.     

Changes in the Sugar Content of the Leaf Sheath of Dwarf Maize Seedlings Associated with Gibberellin-Induced Growth

Changes in the time in sugar and starch content of the firstleaf sheath were compared among normal, d₅, and GA₁-treated(1µg/plant) d₅ seedlings of Zea mays L., from which endospermshad been removed. The patterns of changes in the levels of thesecarbohydrates were more or less similar to one another amongthe three groups of seedlings. Generally, the levels decreasedtoward the time of maturation of the leaf sheath except forthe level of sucrose which showed a sharp increase followedby a decrease. There was an inverse relationship over time betweengrowth and levels of glucose and fructose. Although treatmentwith GA₁ resulted in a decrease in sucrose content, it had practicallyno significant effect on the levels of the other sugars. Inthe leaf sheath of d₅, growth is probably not limited by thelevels of these sugars, and GA-induced elongation does not seemto have any significant correlation with the changes in thelevels of sugars. ¹Present address: Department of Botany, Suri Vidyasagar College,Suri, Dt. Birbhum, West Bengal, India. (Received March 18, 1988; Accepted November 21, 1988)     

Genotypic variation in the responsiveness to GA3 within tall (rht1) and semi-dwarf (Rht1) spring wheat

The effect of GA₃ on coleoptile-and first leaf elongation of tall (rht1) and semi-dwarf (Rht1) nearly-isogenic genotypes, within each of 25 random F₉ wheat families, was determined on seedlings grown in a growth room at 18 °C. Conspicuous and very significant inter-family variation in the response of the first leaf to GA₃ application was found in both the rht1 and Rht1 genotypes. The magnitudes of the response of the different families within genotypes to GA₃ were not related to the leaf length of their untreated seedlings. It is suggested that, under given environmental conditions, background genotypic effects, inducing inter-family variation in responsiveness to GA₃, regulate the elongation growth up to the limits set by the Rht alleles.     

The regulation of leaf elongation and xyloglucan endotransglycosylase by gibberellin in 'Himalaya' barley (Hordeum vulgare L.)

The potential role of xyloglucan endotransglycosylase (XET)in GA-stimulated cell elongation was investigated during leafexpansion in barley (Hordeum vulgare L.). XET activity in aqueousextracts of leaves was detected in all segments along the elongatingblade of leaf 1 of seedlings, but was at highest levels in basalsegments. Leaf 1 elongation rates of gibberellin (GA)-responsivedwarf mutants were lower than the wild type, and accompaniedby reduced levels of XET activity. Leaf elongation rates ofthe dwarfs increased following treatment with gibberellic acid(GA₃) associated with higher levels of XET activity. The slendermutant, crossed into a dwarfing background, exhibited high ratesof leaf 1 elongation and high levels of XET activity withoutadded GA₃. The elongation of leaf 3 in a GA-responsive dwarfmutant was also studied. Treatment with GA₃ resulted in bladeand sheath lengths being 5-fold and 7-fold (respectively) thelengths of controls, and again there were increases in bladeand sheath XET activities. To investigate the basis for changesin XET activity levels two XET-related cDNA clones were isolated.RNAs detected by the two clones occurred at the highest levelsin basal segments of rapidly elongating leaves, but they haddifferent distribution patterns along the leaf. Overall, thedata indicate that an XET-like activity is detectable in barleyleaves, that the activity level and related. Key words: Gibberellin (GA), leaf elongation, Hordeum vulgare, xyloglucan endotransglycosylase (XET)     

Abscisic Acid Inhibition of Gibberellic Acid and Cyclic 3',5'-Adenosine Monophosphate Induced α-Amylase Synthesis

The induction of α-amylase synthesis in barley aleurone by cyclic 3′,5′-adenosine monophosphate or GA₃ was inhibited by abscisic acid. The concentration of ABA required to inhibit α-amylase induction by the cyclic nucleotide in the extract was one-fiftieth to one hundredth of that required for GA₃-induced α-amylase. It is concluded that the effects of ABA on GA₃ and cyclic nucleotide induced α-amylase synthesis in the aleurone are independent and indirect.     

Differential rates of α-amylase and acid phosphatase induction by Ga3 in embryoless half-seeds and isolated aleurones of wheat

The induction of α-amylase and acid phosphatase by gibberellic acid (GA₃) was significantly higher (2–4-fold) in embryoless half-seeds of wheat than that observed in the excised aleurones. Addition of endosperm extract to excised aleurones enhanced the stimulatory effect of GA₃ on amylase activity by approximately 2-fold. Substitution of endosperm extract by 19 amino acids in GA₃-treated aleurones also brought about a 2–2.5-fold stimulation of α-amylase activity. Subsequent studies revealed that the addition of seven non-polar amino acids (0.5 mM each) was sufficient for the enhanced induction of α-amylase (1.8–2.5-fold) in GA₃-treated aleurones. A similatory effect of endosperm extract and amino acids on acid phosphatase activity was observed in GA₃-treated wheat aleurones. These observations are of physiological significance since an increased pool of free amino acids (5-fold) was also witnessed in the incubation medium of GA₃-treated half-seeds in comparison to the hormone-treated aleurones. The relative abundance of free amino acids in half-seed seems vital for the maximal induction of α-amylase and acid phosphatase. Thus, the presence of endosperm tissue associated with the aleurone layers is crucial for enhanced rate of production of GA₃-induced α-amylase and acid phosphatase in the wheat system.     

Regulation of phenylalanine ammonia-lyase activity and growth in lettuce by light and gibberellic acid

Abstract The effects of light and gibberellic acid (GA3) on growth and phenylalanine ammonia-lyase (PAL) activity were studied in seedlings of lettuce (Lactuca sativa L.). Using an in vivo assay for PAL it was shown that wounding caused by excising hypocotyls results in an increase in PAL activity with time that can mask the effect of light on the activity of this enzyme. When hypocotyl sections were excised from light-treated seedlings immediately prior to the in vivo assay of PAL, light was shown to cause a marked increase in PAL activity. Experiments with an inhibitor of PAL activity, α-aminooxy-β-phenylpropionic acid (AOPP), confirmed that the volatile radioactive products measured in the in vivo assay resulted from the activity of PAL. Gibberellic acid suppresses the light-induced increase in PAL activity and there is an inverse relationship between GA₃-induced growth and the activity of PAL. Over a wide range of GA₃ concentrations, the activity of PAL is also inversely correlated with growth rate along the length of the hypocotyl section; the upper halves of sections elongate more rapidly and have lower levels of PAL than the lower halves. Despite the strong correlation between growth and PAL activity, experiments with AOPP and t-cinnamic acid show that it is unlikely that elongation is regulated directly by products of PAL activity.     

Gibberellic-acid-induced cell elongation in pea epicotyls: Effect on polyploidy and DNA content

In gibberellic-acid(GA₃)-treated epicotyls of dwarf peas (Pisum sativum L.) grown in the light, DNA (per cell and per epicotyl) is followed. Histofluorometric DNA determinations show that GA₃-promoted cell elongation is not accompanied by increased endomitosis, but chemical estimations show an increased DNA content per epicotyl. This difference must therefore be the result of increased mitotic activity in the GA₃-treated tissue. Epicotyls of seedlings grown with or without cotyledons under continuous light with GA₃ are tetraploid, as are those of ecotylized embryos grown in darkness. These epicotyls reach no more than half the length of octaploid epicotyls of seedlings grown in darkness. This result provides evidence for a relationship between polyploidy and final possible cell length.     

Gibberellins and Heterosis in Maize : II. Response to Gibberellic Acid and Metabolism of [H]Gibberellin A(20)

Two maize inbreds, CM7 and CM49, and CM7 × CM49, their F₁ hybrid (which displayed significant heterosis), were examined with regard to response to exogenous gibberellin A₃ (GA₃), and in their ability to metabolize GA₂₀, a native GA of maize. The leaf sheath elongation response to GA₃ was far greater for the imbreds than for their hybrid. The inbreds also displayed significant elongation of the leaf blades in response to GA₃, whereas the hybrid was unaffected. Promotion of cell division in the leaf sheath of CM7 and the hybrid was effected by GA₃, but no promotion of cell elongation was observed in CM49, even though significant leaf sheath elongation occurred. Shoot dry weight of both inbreds was significantly increased by GA₃, but response by the hybrid in this parameter was slight and variable. Root dry weight of CM7 was significantly increased by GA₃, but was unchanged in CM49 and the hybrid. Thus, inbred shoot dry weight increases effected by GA₃ were not at the expense of the root system. Rapid metabolism of [2,3-³H]GA₂₀ occurred in all genotypes, although genotypic differences were observed. The hybrid had the highest rates of metabolism to GA glucosyl conjugate-like substances. Oxidative metabolism was also fastest in the hybrid, followed by CM7, and slowest in CM49, the slowest-growing inbred. Thus, rate of GA₂₀ metabolism is under genetic control in normal (i.e. not dwarfed) maize genotypes. These results, taken together with previous reports that the hybrid has significantly enhanced levels of endogenous GA-like substances, suggest that GA play a role in the expression of heterosis in maize.     

Invertase activity and cell growth in lentil epicotyls

The activity of invertase and its relation to growth were studied in the epicotyls of lentil seedlings incubated in the presence and absence of gibberellic acid (GA₃).

Invertase activity per epicotyl increases relatively more rapidly than does length, reaches a maximum during most active elongation, and declines upon cessation of growth.

GA₃ enhances both growth and increase in invertase activity, without altering the kinetics of the 2 processes. If GA₃ is added during incubation invertase activity increases more rapidly than does elongation rate.

Incubation of the seedlings in solutions of polyethyleneglycol inhibits the increase of both growth and invertase activity, the latter actually undergoing a decline, but causes no great change in the relative effect of GA₃. In presence of polyethyleneglycol GA₃ has however a relatively greater effect on invertase activity than on growth.

Sugars in the incubation medium have no significant effect on growth and invertase activity in the epicotyl, except inhibition at relatively high concentrations.

Cycloheximide, actinomycin D, and 5-fluorodeoxyuridine (FUDR) inhibit both growth and the increase in invertase activity. Added during incubation cycloheximide causes complete inhibition of growth and a decrease in invertase activity with no appreciable lag phase. With actinomycin D and FUDR the inhibition occurs after lag periods of 2 to 3 and of at least 10 hours, respectively. Thus the increase in enzyme activity is very probably based on de-novo synthesis, and the enzyme is in a state of turnover during growth.

The enzyme is present in soluble form in the cytoplasm, not firmly bound to any cell structures.

     

Growth Responses and Allocation of Assimilates of Rice Seedlings by Paclobutrazol and Gibberellin Treatment

Paclobutrazol [(2RS,3RS)-1-(4-chlorophenyl)methyl-4,4-dimethyl-2-(1h-1,2,4-trizol-1-yl)penten-3-ol] effectively decreased vegetative growth of rice (Oryza sativa L.) seedlings and increased the chlorophyll content. The number of veins in a leaf, the calculated number of stomata per leaf, and the length of guard cells were not altered by the paclobutrazol treatment, suggesting an effect on cell elongation. The allocation pattern of carbohydrates was changed by either gibberellin (GA) or paclobutrazol treatment. GA₃ induced more shoot growth and less accumulation of starch than the control and paclobutrazol-treated seedlings. Photosynthetic ability was not affected by either paclobutrazol or GA₃ treatment. Paclobutrazol-treated plants allocated a smaller amount of photosynthates for vegetative shoot growth and stored more as starch in the crowns than the control and GA₃-treated plants. The same starch degrading activity in the crown tissue of paclobutrazol-treated seedlings as in control plants suggests that the accumulated starch is utilized in a normal activity for growth including leaf emergence, tiller formation, and root production, resulting in improved seedling quality. Received May 30, 1996; accepted December 10, 1996     

Excess nickel modulates activities of carbohydrate metabolizing enzymes and induces accumulation of sugars by upregulating acid invertase and sucrose synthase in rice seedlings

The effects of increasing concentrations of nickel sulfate, NiSO₄ (200 and 400 μM) in the growth medium on the content of starch and sugars and activity levels of enzymes involved in starch and sugar metabolism were examined in seedlings of the two Indica rice cvs. Malviya-36 and Pant-12. During a 5–20 day growth period of seedlings in sand cultures, with Ni treatment, no definite pattern of alteration in starch level could be observed in the seedlings. In both roots and shoots of the seedlings Ni treatment led to a significant decrease in activities of starch degrading enzymes α-amylase, β-amylase, whereas starch phosphorylase activity increased. The contents of reducing, non-reducing, and total sugars increased in Ni-treated rice seedlings with a concomitant increase in the activities of sucrose degrading enzymes acid invertase and sucrose synthase. However, the activity of sucrose synthesizing enzyme sucrose phosphate synthase declined. These results suggest that Ni toxicity in rice seedlings causes marked perturbation in metabolism of carbohydrates leading to increased accumulation of soluble sugars. Such perturbation could serve as a limiting factor for growth of rice seedlings in Ni polluted environments and accumulating soluble sugars could serve as compatible solutes in the cells under Ni toxicity conditions.