Correlative Studies on Plant Growth and Metabolism. III. Metabolic Changes Accompanying Inhibition of the Longitudinal Growth of Stem and Root by Kinetin

Kinetin-induced expansion of lettuce (Lactuca sativa) cotyledons and inhibition of root are accompanied by parallel changes in protein nitrogen. However, during its inhibition of the longitudinal growth and water uptake of hypocotyl and pea (Pisum sativum) epicotyl sections kinetin markedly stimulates protein synthesis. Kinetin seems to separate auxin induced effects on protein synthesis and water uptake and indicates that water uptake and protein synthesis may not necessarily be correlated.

In contrast to gibberellic acid, kinetin restricts in lettuce seedlings, the mobilization of nitrogen reserves from the cotyledons, and kinetin induced growth is accompanied by a high protein nitrogen/soluble-nitrogen ratio which is characteristic of growth in light. Growth in light may be under the dominant control of kinins.

     

The effect of phytochrome and white light of high and low intensity on the uptake and distribution of 14C-labelled kinetin in radish seedlings (Raphanus sativus)

The influence of light intensity and phytochrome on the uptake of ¹⁴C-kinetin (6-furfurylamino-[8- ¹⁴C]-purine) by the plant and the translocation of the phytochrome between the roots, the hypocotyl and the cotyledons were investigated with radish seedlings ( Raphanus sativus L. cv. Saxa Treib) grown in the dark or under white light of high (20,000 lux, 90 W m⁻²) or low intensity (2,000 lux, 14 W m⁻²). The highest uptake of labelled kinetin was found in plants grown in continuous darkness. The total uptake of kinetin was decreased by strong light and to a finally higher extent by weak light. Under white light most of the kinetin accumulated in the root, whereas in the dark an enhanced translocation of the phytohormone into the cotyledons was observed. In etiolated radish seedlings, light acting on phytochrome (daily 5 min red or far red light pulses) decreased the translocation of ¹⁴C-kinetin into the cotyledons. Under far red light a pronounced uptake of the phytohormone into the roots was found. The data are discussed with regard to the interaction of light and phytohormones on plant development.     

Changes in a Leaf-growth Substance in Cotyledons and Primary Leaves during the Growth of Dwarf Bean Seedlings

The growth of etiolated dwarf bean leaf disks in darkness wasused to assay leaf-growth substances extracted from plant tissuesand separated on chromato-grams. A leaf-growth substance, with the same Rf value in isopropanol/ammoniaas gibberellic acid, was found in the acidic fraction of extractsfrom dwarf bean primary leaves and cotyledons. Neither neutralnor basic leaf-growth substances were found in primary leafextracts. The amount of leaf-growth substance per cotyledon reached amaximum at the 4th day in cotyledons, whether bean plants weregrown in the light or dark. Primary leaves of dwarf bean plantsgrown in the light reached their maximum content of leaf-growthsubstance later than the cotyledons; the time of maximum contentcoincided with the maximum growth-rate of the primary leaves.Leaf-growth substances did not accumulate in primary leavesof plants grown in the dark.     

Allelopathy in Heracleum laciniatum: Inhibition of Lettuce Seed Germination and Root Growth

Both dark and red light germination of lettuce seeds (cv. “Maikönig”) as well as their root and hypocotol elongation were inhibited when the seeds were sown in petri dishes together with a few seeds of Heracleum laciniatum Horn. This inhibition was not significantly counteracted by the presence of gibberellic acid (GA₃) or/and 6-benzylaminopurine (BA). However, a large proportion of the lettuce seeds germinated abnormally (only cotyledons emerged) when treated with BA in the presence of Heracleum seeds. GA₃ had alone no significant effect on abnormal germination, but it counteracted the effect of BA to some extent. The inhibitory effect of Heracleum seeds gradually disappeared during a moist incubation period of one to seven days in darkness at 25°C. When lettuce seeds were pre-incubated together with Heracleum seeds for one to five days the remaining, non-germinated lettuce seeds had lost their ability for subsequent germination in darkness in distilled water. This induced dark dormancy was to a great extent broken by red light, but not by GA₃ or/and BA. H. laciniatum seeds inhibited the germination of Salix pentandra seeds and to some extent also the germination of radish but had no effect on the germination of spruce.     

Gibberellic Acid-Promoted Lignification and Phenylalanine Ammonia-lyase Activity in a Dwarf Pea (Pisum sativum)

The effects of gibberellic acid on lignification in seedlings of a dwarf and a tall cultivar of pea (Pisum sativum) grown under red or white light or in the darkness, were studied. Gibberellic acid (10⁻⁶-10⁻⁴ m) promoted stem elongation in both light and dark and increased the percentage of lignin in the stems of the light-grown dwarf pea. The gibberellin had no effect on the lignin content of the tall pea although high concentrations (10⁻⁴ m) promoted growth of the tall plants. Time course studies indicated that the enhanced lignification in the gibberellin-treated dwarf plants occurred only after a lag period of several days. It was concluded that gibberellic acid-enhanced ligmification had no direct relation to gibberellic acid-promoted growth. The activity of phenylalanine ammonia-lyase (E.C. 4.3.1.5) was higher in gibberellin-treated dwarf plants grown under white or red light than in untreated dwarf plants. Gibberellic acid had no detectable effect on the activity of this enzyme when the plants were grown in darkness, just as it had no effect on lignification under dark conditions. The data suggest that in gibberellin-deficient peas the activity of phenylalanine ammonia-lyase is one of the limiting factors in lignification.     

Chlorophyll accumulation in cotyledons, hypocotyls and primary needles of Pinus pinea seedlings in light and dark

Seeds of Pinus pinea L. were germinated and grown in darkness or under natural photoperiodic (day/night) conditions for 8 weeks. Samples were taken for analysis at frequent intervals and chlorophyll (Chl) and protochlorophyllide (Pchlide) contents of cotyledons, hypocotyls and primary needles determined spectrophotometrically alter separating the esterified from unesterified pigments by partitioning between ammoniacal acetone and petroleum ether. Chlorophyll accumulated in cotyledons, hypocotyls and primary needles of dark as Well as light grown seedlings. The maximum Chl concentrations achieved in darkness in these organs were approximately 60, 20 and 10% respectively, of concentrations achieved in light. The tinsel of rapid Chl accumulation by cotyledons in darkness coincided with the emergence of the radicle from the seed. Light had no effect on the timing of this process or on the duration of the lag period. However, it extended the exponential growth phase of cotyledons by at least a week and. once the seedlings were about 2 weeks old, enabled Chl to accumulate at a faster rate. Time courses of Chi and Pchlide accumulation by cotyledons in darkness were similar. Pchlide did not accumulate prior to the onset of Chi accumulation and Chl slopped accumulating once the Pchlide pool reached maximum size. The implications of these findings are discussed and a hypothesis to account for the inability of primary needles of smaller seeded pines to accumulate Chl in darkness is proposed.     

Phytochromabhängige Veränderungen des Wachstums und der Ionenaufnahme etiolierter Erbsenkeimlinge

Summary Inhibition of internodial growth of pea seedlings by light is compensated for by increased growth of leaves. At a given time the sum of fresh weight of internodes plus the product of fresh weight of leaves times a certain factor is constant in darkness or with different periods of light. This correlation may reflect a competition of internodes and leaves for materials delivered at a lightindependent rate from the cotyledons. This hypothesis was tested by immersing roots of pea seedlings into ⁸⁶Rb labelled K-solutions for one day in darkness, removing the plants from the solutions, exposing the seedlings to near or far red light and measuring the radioactivity and fresh weights of leaves and internodes separately. Radioactivity and fresh-weight were both dependent on phytochrome; i.e. inhibition of ion uptake and of growth in internodes and promotion of both processes in leaves by near red light as compared to dark or far red controls are mediated by phytochrome.Short time experiments of ion uptake by the roots show that K transport into the shoot organs is promoted by light after a lag phase of somewhat more than one hour. This interval corresponds well to the lag phase of the light induced growth inhibition of internodes.Seedlings deprived of cotyledons and roots grow well in water but exhibit no difference in growth rate of leaves and internodes in light and darkness. Light dependence is restored if the seedlings are submersed in approximately 3% sucrose solutions. This result seems to indicate that the influence of light on growth rates of leaves and internodes is dependent on the uptake of material by the cell. It seems possible that in the etiolated pea seedling light promotes growth of leaves by promoting uptake and hampers growth of internodes by inhibiting uptake of essential growth material delivered from the cotyledons.     

Isolation and chemical identification of a lettuce cotyledon factor, a synergist of the gibberellin action in inducing lettuce hypocotyl elongation

Removal of cotyledons from intact lettuce seedlings retardedhypocotyle elongation caused by gibberellic acid. This inhibitoryeffect of cotyledon excision was alleviated by incubating decotylizedseedlings with a hot water extarct from excised cotyledons orintact seedlings. This active principle was identified as dihydroconiferylalcohol on the basis of its IR, NMR and mass spectra. (Received August 28, 1973; )     

THE EFFECT OF LIGHT ON THE USE OF THE NITROGEN RESERVES OF GERMINATING SOYBEAN SEEDS

Light affects the mobilization and distribution of several of the storage components of the cotyledons of germinating soybean seeds. The nitrogen content of the cotyledons began to decrease during the first day of germination, continued through day 12 for plants in the light, and day 14 for those in the dark. Cotyledons from both treatments had lost about the same amount of nitrogen by day 14. Plants from both treatments lost about the same amount of dry weight by day 8, but those in the light had taken up nitrogen from the nutrient solution; while those in the dark showed no increase. The plants in the light had higher concentrations of soluble amino nitrogen in the cotyledons than did those in the dark, but the opposite was true for the seedling axis. Aspartate and its amide accounted for half or more of the total free amino acids in all parts of dark-grown plants at 6 and 14 days. In the light-grown plants aspartate and asparagine usually accounted for less than half of the total free amino acids in all plant parts except the cotyledons at 6 and 14 days. Total soluble amino acids were much lower in these plants than those in the dark, excepting the cotyledons.     

Effects of Kinetin, Gibberellins and (±) Abscisic acid on the Germination of Lettuce (Lactuca sativa)

Germination responses of achenes of lettuce (Lactuca sativa L, cv. Arctic King) to treatment with kinetin, gibbe-rellins and abscisic acid were examined over a range of temperatures: in both light and dark. Kinetin (0.1–10 mg/l) strongly promoted germination at temperatures above 27±C in continuous light or after short periods of illumination during the early stages of imbibition. It also relieved the inhibitory affects of abscisic acid in these conditions. In total darkness however kinetin treatment resulted in only a minor promotive effect. Treatment with gibberellic acid (A₃) or a mixture of gibberellins A₄ and A₇ were much less effective in promoting germination at higher temperatures of lettuce achenes exposed to light but were strongly promotive in the dark.     

STUDIES ON THE SOLUBLE CARBOHYDRATES AND CARBOHYDRATE PRECURSORS IN GERMINATING SOYBEAN SEED

The total soluble carbohydrate fraction of the cotyledons and embryo axis of germinating soybean seedlings declined rapidly during the first 3 days of germination. This depletion began earlier in the embryo axis than in the cotyledon. The total carbohydrate content of the cotyledons of plants grown in light and plants grown in dark was approximately the same for the first 7 days of germination. Between day 9 and 13 the total carbohydrate content of the cotyledons of soybean seedlings grown in dark was higher than that of plants grown in light. The reducing sugar content of light-grown soybean cotyledons increased approximately 5-fold during the first 9 days of germination, whereas that of dark-grown soybean cotyledons increased more slowly during this interval. Reducing sugars in the embryo increased during the early stages of germination until they approximately equalled the total carbohydrate. Between day 4 and 13, oil was depleted more rapidly in the cotyledons of seedlings grown in light than those grown in the dark. The reserve carbohydrates of soybean embryos and cotyledons consisted primarily of low molecular weight oligosaccharides, particularly sucrose, stachyose, and raffinose. These compounds decreased rapidly during germination. The isocitritase activity in the cotyledons of germinating soybean seeds increased rapidly for the first 6 days of germination and then decreased for the next 7 days. The isocitritase activity of plants grown in the dark was higher than that of the plants grown in light at all stages of development, particularly between day 7 and 11.     

The flexible interrelation between AOX respiratory pathway and photosynthesis in rice leaves.

Alternative respiratory pathway was investigated in rice seedlings grown under total darkness, light/dark cycle, or continuous light. The capacity of the alternative pathway was relatively higher in leaves that had longer light exposure. An analysis of rice AOX1 multigene family revealed that AOX1c, but not AOX1a and AOX1b, had a light-independent expression. The alternative oxidase (AOX) inhibitor, salicylhydroxamic acid (SHAM, 1mM), inhibited nearly 68% of the capacity of the alternative pathway in leaves grown under different light conditions. The plants grown under different light periods were treated with SHAM and then were exposed to illumination for 4h. The transition from dark to 4h of light stimulated the capacity of alternative pathway in etiolated rice seedlings and in those grown under light/dark cycle, whereas the capacity of the alternative pathway was constant in seedlings grown under continuous light with additional 4h of illumination. Etiolated leaves did not show any CO(2) fixation after 4h of illumination, and the increase in chlorophyll content was delayed by the SHAM pretreatment. When seedlings grown under light/dark cycle were moved from dark and exposed to 4h of light, increases in chlorophyll content and CO(2) fixation rate were reduced by SHAM. Although these parameters were stable in plants grown under continuous light, SHAM decreased CO(2) fixation rate but not the chlorophyll content. These results indicate that the role and regulation of AOX in light are determined by the developmental stage of plant photosynthetic apparatus.     

Requirement of cotyledons for gibberellic acid-induced hypocotyl elongation in lettuce seedlings. Isolation of the cotyledon factor active in enhancing the effect of gibberellic acid

The role of cotyledons in hypocotyl elongation caused by gibberellicacid was studied using young seedlings of lettuce, Lactuca saliva,var. ‘Grand Rapids’. Removal of cotyledons fromintact seedlings resulted in a depression of hypocotyl elongationcaused by gibberellic acid. Gibberellic acid-induced hypocotylelongation in decotylized seedlings, was however, substantiallyenhanced by incubating the seedlings together with excised cotyledons.The exudate from excised cotyledons also enhanced the effectof gibberellic acid on hypocotyl elongation in decotylized seedlings.This active principle (named the cotyledon factor) in the cotyledonexudate was stable against heating at 100?C for 15 min, permeatedthe dialysis membrane, and was extractable with ethyl acetate.Biological activity of the cotyledon factor was not replacedby indole-3-acetic acid, kinetin, cyclic AMP, vitamins, sucroseor inorganic nutrients. The biological significance of the cotyledonfactor is discussed in relation to the action of gibberellicacid. (Received February 14, 1973; )     

Die Hemmung der Isocitrat-Lyase bei Wassermelonenkeimlingen durch Weißlicht

Summary The isocitrate lyase activity (E.C. 4.1.3.1.) from watermelon cotyledons (Citrullus vulgaris Schrad.) is inhibited by white light (Fig. 5). To exclude artefacts during enzyme preparation the following experiments were performed (Table 1 and 2): 1. Mixing of raw extracts from cotyledons of light and dark grown seedlings. 2. Joint homogenization a) of cotyledons from light and dark grown seedlings, b) of purified isocitrate lyase together with cotyledons from light or dark grown seedlings. The total activity corresponded to an amount which was expected for the sum of the individual activities. The results justify the conclusion that the inhibition of the isocitrate lyase by light is real, and that the measured enzyme activities are close to the true enzyme concentrations in the plant tissue. — The relatively slow inhibition of the enzyme activity caused by light seems to be correlated with the formation of the photosynthetic apparatus.     

Actions of gibberellic Acid and phytochrome on the germination of grand rapids lettuce seeds

Red light and gibberellic acid were about equally effective in promoting germination of Grand Rapids lettuce (Lactuca sativa L.) seeds. With initial far red light treatment more than 80% remained dormant in subsequent dark storage. After 2 days of dark storage, red light effectively promoted germination, while gibberellic acid action was weak. With between 2 and 10 days of dark storage, gibberellic acid had little effect, while promotion by red light decreased slowly and finally disappeared. After 10 days of dark storage, both gibberellic acid and red light were required for germination. The dark storage treatment interferes with phytochrome-independent germination processes and cannot be overcome by added gibberellic acid. However, storage may also decrease the effectiveness of endogenous gibberellins. Phytochrome-dependent germination seems to require only low levels of endogenous gibberellin activity or the addition of gibberellic acid. Gibberellins and red light appear to act on germination by regulation of sequential sites of a branched-looped pathway.     

Chlorophyll formation and the development of photosynthesis in dark grown seedlings of Picea abies

Abstract Seeds of Picea abies were germinated and grown in either darkness or constant light. The chlorophyll content and photosynthetic carbon dioxide uptake of developing seedlings of different ages was determined. Ten-day-old dark grown seedlings showed an instant ability for photosynthetic carbon dioxide uptake and also formed further chlorophyll most rapidly upon subsequent illumination. These activities progressively diminished when the dark growth period was extended. Light grown seedlings reached a maximum chlorophyll level after 15 days growth, and this preceded maximal photosynthetic development.     

Photoperiod and light quality effects on rate of dark respiration and on photosynthetic capacity in developing seedlings of Chenopodium rubrum

Development and acclimation of energy transduction were studied in seedlings of Chenopodium rubrum L. ecotype selection 184 (50° 10' N; 105° 35' W) in response to photomorphogenic and photoperiodic treatments. Dark respiration and photosynthetic capacity [nmol O₂ (pair of cotyledons)⁻¹ h⁻¹] were measured with an oxygen electrode. Changes in chloroplast ultrastructure were analyzed concomitantly. After germination, seedlings were grown at constant temperature either in darkness or in continuous light (white, red, far-red and blue) or were subjected to diurnal cycles of light/dark or changes in light quality. Dark respiration was low in far-red light treated seedlings. In red light treated seedlings dark respiration was high and the mean value did not depend on fluence rate or photoperiod. Blue light stimulated transitorily and modulated dark respiration in photoperiodic cycles. Photosynthetic capacity was reduced by far-red light and increased by red light. In response to blue light photosynthetic capacity increased, with indications of a requirement for continuous energy input. Phytochrome and a separate blue light receptor seemed to be involved. In continuous red light a clear cut circadian rhythm of dark respiration was observed. Blue light had a specific effect on chloroplast structure.     

ACTION OF KINETIN ON PHOTOSENSITIVE GERMINATION OF LETTUCE SEED AS COMPARED WITH THAT OF GIBBERELLIC ACID

1. Experiments with the seeds of Grand Rapids lettuce showedthat the germination induced by gibberellic acid or by red lightis strongly accelerated by kinetin, although the latter itselfcan promote the germination in the dark only slightly. The reversiblelight reactions of the phytochrome system interact with kinetintreatment just as effectively as with water-imbibed controls.The site of primary action of red light is not altered by kinetintreatment. Kinetin does not modify the water uptake of the seedsfor at least 8 hours. 2. Kinetin was found to inhibit the growth of the hypocotyland root of the seed, but to promote very markedly the expansionof the cotyledons. This effect was observed not only with cotyledonsin intact seeds but also with isolated cotyledons. The expansionof kinetin-treated cotyledons is further promoted by red light,but not by far-red, as is also the case with germination itself. 3. A number of purine derivatives which have been reported topromote germination also cause expansion of isolated cotyledons. 4. Gibberellic acid promotes both hypocotyl elongation and cotyledonexpansion in the dark, but this effect does not interact withthe phytochrome system. The site of action of gibberellic acidprobably lies in the axis. 5. It is concluded, therefore, that the site of kinetin actionis in the cotyledons, whose expansion helps to break the seed-coatwhen light or gibberellin has contributed the primary stimulus. ¹ Present address: Johnson Foundation for Medical Physics, Universityof Pennsylvania, Philadelphia, Pa. (Received January 16, 1963; )