Extraction of Enzymes from Cell Walls of Sugar Beet Cells Grown in Suspension Culture

Various glycosidases were extracted from cell walls of sugarbeet cells grown in suspension culture using three successiveprocedures employing saline, EDTA, and commercial cellulase.Saline was effective for extracting acid invertase, ß-galactosidase,and ß-glucosidase. EDTA extracted most of the -galactosidaseand some of the ß-glucosidase. It was most effectivebetween 27?C and 40?C. Commercial cellulase could extract mostof the -mannosidase in the cell wall when used at 27?C for 96h. These three procedures could not extract some of the acidinvertase and ß-glucosidase. The results suggest thatthe cell wall glycosidases are associated with different polysaccharides. These extraction procedures were also applied to the cell wallsof intact tissues, such as cotyledons, hypocotyls plus roots,and mature roots of sugar beets. EDTA as well as saline wasquite effective for extracting bound enzymes from the cell wallof intact tissue, which indicated that extraction with EDTAis useful for liberating bound enzymes from plant cell walls. (Received October 19, 1987; Accepted March 16, 1988)     

Comparative effects of gibberellic acid and N6-benzyladenine on dry matter partitioning and osmotic and water potentials in seedling organs of dwarf watermelon

Apical applications of 0.2 μg N⁶-benzyladenine (BA), a synthetic cytokinin, or 5 μg of gibberellic acid (GA₃) significantly enhanced hypocotyl elongation in intact dwarf watermelon seedlings over a 48-h period. Accompanying the increase in hypocotyl length was marked expansion of cotyledons in BA-treated seedlings and inhibition of root growth by both compounds. A study on dry matter partitioning indicated that both growth regulators caused a preferential accumulation of dry matter in hypocotyls at the expense of the roots; however, GA₃ elicited a more rapid and greater change than did BA. In comparison to untreated seedlings, BA decreased total translocation of metabolites out of the cotyledons. Water potentials of cotyledons and hypocotyls were determined by allowing organs to equilibrate for 2 h in serial concentrations of polyethylene glycol 4000. Osmotic potentials were determined by thermocouple psychrometry. During periods of rapid growth in cotyledons and hypocotyls of BA-treated seedlings and in hypocotyls of GA-treated seedlings, the osmotic potential increased and the turgor pressure decreased in relation to untreated seedlings, indicating that cell wall extensibility was being increased. Osmotic potentials were lower in hypocotyls of GA-treated than in those of BA-treated seedlings, even though growth rates were higher in GA-treated seedlings, indicating that the latter treatment was generating more osmotically active solutes in hypocotyls.     

Promotion of Hypocotyl Elongation in Watermelc Seedlings by 6-Benzyladenine

Hypocotyl elongation under white fluorescent light was aboutdoubled in dwarf watermelon (Citrullus lanatus0 (Thunb.) Matsu.and Nakai) seedlings treated with 0.1 to 0.3 µg apicaland 3 x 10–6 to 10.3 M root applications of 6-benzyladenine(BA). BA-enhancement of growth occurred primarily during thefirst 48 h after treatment. Increased hypocotyl length in BA-treatedseedlings was attributed more to longer cells than to an increasein cell number. Early hypocotyl growth of normal seedlings wasalso significantly enhanced by BA although final hypocotyl lengthwas not substantially affected. Benzyladenine caused expansion of cotyledons and, at higherdoses, lateral expansion of hypocotyls. BA-induced increasesin fresh weight of cotyledons and hypocotyls were accompaniedby an increase in dry weight of hypocotyls at the expense ofroots which had less dry matter than untreated seedlings.     

Comparative effects of gibberellic acid and N6-benzyladenine on dry matter partitioning and osmotic and water potentials in seedling organs of dwarf watermelon

Apical applications of 0.2 g N⁶-benzyladenine (BA), a synthetic cytokinin, or 5 g of gibberellic acid (GA₃) significantly enhanced hypocotyl elongation in intact dwarf watermelon seedlings over a 48-h period. Accompanying the increase in hypocotyl length was marked expansion of cotyledons in BA-treated seedlings and inhibition of root growth by both compounds. A study on dry matter partitioning indicated that both growth regulators caused a preferential accumulation of dry matter in hypocotyls at the expense of the roots; however, GA₃ elicited a more rapid and greater change than did BA. In comparison to untreated seedlings, BA decreased total translocation of metabolites out of the cotyledons. Water potentials of cotyledons and hypocotyls were determined by allowing organs to equilibrate for 2 h in serial concentrations of polyethylene glycol 4000. Osmotic potentials were determined by thermocouple psychrometry. During periods of rapid growth in cotyledons and hypocotyls of BA-treated seedlings and in hypocotyls of GA-treated seedlings, the osmotic potential increased and the turgor pressure decreased in relation to untreated seedlings, indicating that cell wall extensibility was being increased. Osmotic potentials were lower in hypocotyls of GA-treated than in those of BA-treated seedlings, even though growth rates were higher in GA-treated seedlings, indicating that the latter treatment was generating more osmotically active solutes in hypocotyls.Scientific Contribution No. 1219 from the New Hampshire Agricultural Experiment Station.     

Phytochrome controlled acid RNase: An “attached” protein of ribosomes

The light-dependent increment in RNase activity (which is ribosome bound in cell extracts) is distributed as a gradient increasing from base to hook of lupin hypocotyls. No evidence was found of non-specific or of specific activation of pre-formed enzyme molecules following isolation, either before or after (latent activity) destruction of particles. The autodegradation capacity of ribosomes isolated from irradiated cells was almost double that of ribosomes from etiolated tissue. It is proposed that association between the bulk of the light-controlled RNase fraction and lupin ribosomes results from binding of soluble protein. It is not clear whether binding is specific or an artifact of isolation.     

Hormonal Regulation of Phototropism in the Light-grown Sunflower Seedling, Helianthus annuus L.: Immobility of Endogenous Indoleacetic Acid and Inhibition of Hypocotyl Growth by Illuminated Cotyledons

Light-grown sunflower seedlings contain 10^–7–3 x10^–7 M indoleacetic acid (IAA). The even distributionof this endogenous IAA in straight hypocotyls does not changeduring phototropic curvature. The diffusate from hypocotylscontains substance(s) influencing the elongation rate of Avenacoleoptile segments but hardly any IAA. Phototropic curvature of the hypocotyl requires the presenceof illuminated cotyledons. Illumination of cotyledons inhibitshypocotyl growth. It is concluded that the phototropic response of the sunflowerhypocotyl is regulated by factors promoting and inhibiting cellelongation other than IAA.     

Culture of Cotyledon and Hypocotyl Protoplasts from True Potato Seedlings in Solanum tuberosum L.

Culture of protoplast using cotyledon and hypocotyl as the donor tissue from true potato seedlings (TPSs) of 3 breeding lines (DTO-33, ND 860-2 and BN 9815-3) of Solanum tuberosum L. was studied. The cotyledons and hypocotyls of TPSs just extended were excised and digested in an enzyme solution containing 1 % cellulase and 0. 5 % macerozyme for 17—20 h after vacuum infiltration of the tissue in the solution. The protoplasts were cultured in an improved liquid medium and transferred onto solid media for callus culture and shoot regeneration. Some factors affecting the efficiency of cotyledon and hypocotyl protoplast culture were studied. The results showed that using the cotyledons and hypocotyls as donor tissues for protoplast isolation and culture in potato, the division frequency of protoplast derived cells was significantly higher than that using the leaves and shoot-tips of the test-tube plantlets: the yield and quality of the protoplast from TPSs cultured under continuous high light intensity (3000 Ix) were much higher than the TPSs cultured under low light intensity (1000 Ix), and no intact protoplast was ever obtained from the TPSs cultured in continuous dark condition. Vacuum infiltration of the cotyledon and hypocotyl segments in enzyme solution before digestion increased protoplast yield. The yield of protoplasts from hypocotyl tissue was significantly higher than from the cotyledon, but there was no significant difference in quality between the protoplast derived from the two tissues. The significance, advantages and shortcomings of using the cotyledons and hypocotyls as the donor tissues for isolation and culture of potato protoplasts are dicussed.     

The effect of cotyledons on hypocotyl elongation in light-grown bean seedlings: Comparison between dwarf and tall varieties

Hypocotyl sections with and without the cotyledons were cutfrom bean seedlings and incubated under white light of 6000lux. The cotyledons had an inhibitory effect as well as a promotiveeffect on hypocotyl growth. The former effect was more strikingin the dwarf variety, and the latter in the tall variety. Whenthe hypocotyl units were exposed to light for shorter times(6 hr or less) or incubated under weaker light (1600 and 50lux), the inhibitory effect of the cotyledons decreased greatly,and in the tall variety the presence of cotyledons producedno inhibition, but a promotion of hypocotyl growth. GA treatmentenhanced hypocotyl growth and counteracted the growth inhibitioncaused by the cotyledons. On the whole, the GA effect was moremarked in the tall variety than in the dwarf. The elongation of bean hypocotyls may be controlled by a balancebetween the inhibitory and promotive effects of cotyledons,and the predominance of the former over the latter may be oneof the causes for expressing dwarfing. (Received November 13, 1976; )     

Natural growth inhibitors associated with the cotyledon effect in light-grown dwarf and tall beans

The contents of non-acidic, acidic and bound growth inhibitorsin hypocotyls and cotyledons were compared between dark- andlight-grown dwarf and tall beans by means of thin-layer chromatographyand bioassay. In the non-acidic fraction, one major inhibitoryactivity appeared on the chromatogram, but its Rf zone was differentbetween hypocotyls and cotyledons. In both the acidic and boundinhibitor fractions, one major inhibitory activity appearedat the Rf zone corresponding to ABA. The ABAIike substance whichwas the major inhibitor in the hypocotyl was more abundant inlight-gorwn than dark-grown ones, especially in the dwarf variety,but light irradiation did not cause its transport from cotyledonsto the hypocotyl. A larger amount of bound ABA-like substance,which was the major inhibitor in the cotyledon, was presentin the dwarf than the tall variety regardless of the light condition.Cotyldon-enhanced photoinhibition of hypocotyl growth couldnot be explained by the levels of the xanthoxinand ABA-likeinhibitors, or the transport of these free inhibitors from cotyledons. (Received September 8, 1977; )     

Uptake, transport and metabolism of 14C-2,4-dichlorophenoxyacetic acid (14C-2,4-D) in cucumber (Cucumis sativus L.) explants

The uptake, distribution and metabolism of 2,4-D using 14C-labelled 2,4-dichlorophenoxyacetic acid (14C-2,4-D) was studied in isolated hypocotyl and cotyledon explants of cucumber (Cucumis sativus L.) in vitro. Cotyledons had a higher uptake capacity than hypocotyls; the uptake in cotyledons increased linearly up to 20 h, while in hypocotyls only up to 8 h. The distribution of 14C-activity in both organs was basipetal, but more pronounced in cotyledons. The 2,4-D taken up by cotyledons was metabolized very rapidly: only 7% of 14C-activity was associated with free 2,4-D after 20 h exposure. Hypocotyls metabolized 2,4-D more slowly: after 20 h 50% of the 14C-activity was still associated with free 2,4-D. After short incubation periods (2–5 h) 2,4-D-aspartate was a predominant metabolite, after longer incubation (8–20 h) 2,4-D-glucosyl ester prevailed. Roots or callus were formed on the base of cotyledons depending on the length of exposure to 2,4-D.     

In vitro plant regeneration via organogenesis of cowpea [Vigna unguiculata (L.) Walp.]

Shoot regeneration via organogenesis was achieved from axenic cowpea [Vigna unguiculata subsp. unguiculata L. (Walp.) Verde.] hypocotyls and cotyledons of advanced breeding lines and varieties. Cotyledons and embryos were excised from green immature pods. The apical parts of the embryos were removed and the hypocotyls were transferred to regeneration media. Cotyledons and hypocotyls were tested on media with gradients of several hormonal and putrescine combinations. Cowpea cotyledons and hypocotyls exhibited a pattern of shoot formation that occurred in three distinct phases. Multiple shoots developed within 45 days from the wounded region of the primary hypocotyl and cotyledons in different media containing a high cytokinin concentration. The induced plant explants were then grown for 20 days in low-intensity light (10 μmol m^–2 s^–1) on the same medium and numerous shoot buds emerged de novo from the upper part of the hypocotyl and the wounded part of the cotyledons. These buds had no apparent vascular connection with the parent tissues. The plant regeneration capability of this procedure was tested with several cowpea genotypes, five of which (83D-442, 86D-1010, 93K-624, Vita 3 and Ife Brown) responded positively with shoot development and were able to form roots and whole plants. Some somaclonal variation was observed. Received: 14 June 1996 / Revision received: 14 December 1996 / Accepted: 25 January 1997     

A STARCH-AGAR GEL METHOD FOR THE LOCALIZATION OF STARCH HYDROLYZING ENZYMES IN THE COTYLEDONS AND HYPOCOTYLS OF BEANS

A starch-agar gel assay was developed for the localization of enzyme activity in the cotyledons and hypocotyls of beans. The four major starch hydrolyzing enzymes, α-amylase, β-amylase, debranching enzyme and phosphorylase, were assayed with this technique using gels containing different substrates and buffers. Hypocotyls and cotyledons were sectioned by hand and the sections were incubated on the gel for varying lengths of time. Only α-amylase was observed to increase significantly. While virtually absent in ungerminated axes and hypocotyls from young seedlings, it increased first in the basal region and subsequently in progressively higher portions of the hypocotyl. High activities were first observed in the pith and later in the cortex. The increased activity was most obviously associated with the degradation of the pith, suggesting a regulation relative to cellular autolysis.     

Effect of morphactin on peroxidases and its relationship to rooting hypocotyl cuttings of Impatiens balsamina

Morphactin inhibited the production of roots on hypocotyl cuttingsof Impatiens balsamina with intact apex and cotyledons. Rootswere, however, initiated when cuttings were transferred to water.A new peroxidase isoenzyme in the cotyledons and two in thehypocotyls that were absent in controls, appeared in morphactin-treatedcuttings. However, one isoenzyme in the cotyledons and threein the hypocotyls that were absent in morphactin-treated cuttings,were detected in the controls. These isoenzymes may be involvedin the initiation and development of roots. (Received August 15, 1972; )     

3-Dehydroshikimate hydro-lyase ofPhaseolus mungo seedlings

3-Dehydroshikimate hydro-lyase was extracted and partially purified by ammonium sulfate fractionation from the hypocotyls of etiolatedPhaseolus mungo seedings. The enzyme was most active at pH 7.0 and the Km for DHS was 1.4 mM. Enzyme activity was inhibited byp-chloromercuribenzoate and arsenite, the inhibition being reversed by reduced glutathione. Enzyme activity was found in extracts from young hypocotyls and cotyledons, especially from 5–6 day-old hypocotyls and 3–4 day-old cotyledons. Changes in enzyme activity in both cotyledons and hypocotyls were measured during the early stage of germination. Participation of this enzyme in the catabolism of alicyclic acids is suggested.     

Some Morphogenic Differences between Monoecious and Gynoecious Cucumber Seedlings as Related to Ethylene Production

Exposure of gibberellic acid-treated seedlings of a monoecious cucumber cultivar `Chipper' (Cucumis sativus L.) to ethylene caused thickening of the hypocotyl, inhibited longitudinal growth, and had no effect on fresh weight. Downward curvature of cotyledons was increased by the presence of ethylene. A gynoecious breeding line, `Gy 3,' had thicker hypocotyls and displayed its cotyledons in a more downward position than `Chipper'. Excised hypocotyls of the gynoecious seedlings produced three times as much ethylene as did the monoecious Chipper hypocotyls. Thus, ethylene may play a role in the regulation of cucumber seedling morphology.     

Photocontrol of anthocyanin formation in turnip seedlings

Summary As measured by in vivo spectrophotometry the phytochrome content in etiolated turnip seedlings was higher in cotyledons than in hypocotyls; in the latter, it is confined to the apical part. During early growth in darkness the amount increased in both tissues to a maximum, reached about 40 hours after sowing; the levels then gradually declined. Separation of seedlings into hypocotyl and cotyledons increased the rate of phytochrome loss in the former, but not in the latter.Following 5 minutes of red light P _frdecayed very rapidly in darkness; after 1.5 hours all of the phytochrome was present as P _r, which was presumably not converted initially. In continuous red light the total phytochrome was reduced to below the detection level within 3 hours. Seedling age markedly affected the loss of phytochrome following red light; more was destroyed in older than in younger hypocotyls and apparent new synthesis occurred only in young seedlings. The capacity to synthesise phytochrome differed in cotyledons and hypocotyl. In cotyledons, synthesis occurred following shots of red light varying from 10 seconds, to 6×I minute, but the amount of newly formed phytochrome was not related to the amount destroyed: after 5 hours of continuous red light no new synthesis occurred. In hypocotyls, the amount of phytochrome synthesised was related to the amount previously destroyed, and the phytochrome content after 24 hours of darkness was similar following all red light treatments of 1 minute or longer: new synthesis occurred following 5 hours of continuous red light.In far-red light phytochrome decayed very slowly, approaching the limit of detection after 48 hours. In cotyledons some loss was already observed after 5 hours of far-red and, in hypocotyls, after about 10 hours.These results are discussed in relation to the possible role of phytochrome as the pigment mediating anthocyanin synthesis in prolonged far-red light.     

Glycosidases and acid-invertase activities are not correlated with Phaseolus hypocotyl growth

Changes in α-galactosidase, β-galactosidase, β-glucosidase and acid invertase activities were examined in Phaseolus vulgaris hypocotyls treated with gibberellic acid (GA), naphthyl acetic acid (NAA) and distilled water (DW) (control) in light condition. The activities were estimated both in cytoplasmic and ionically wall-bound fraction. The upper segment showed considerable elongation growth while there was hardly any growth in lower segment. GA and NAA showed distinct promotion and inhibition respectively in hypocotyl growth in upper segment. The glycosidase activities were detected in both the fractions but the activity was more pronounced in cytoplasmic than in wall fraction. Acid invertase activity was present only in cytoplasmic fraction. In lower segment, in both cytoplasmic and wall fraction, the glycosidase activity, in general, showed a decreasing trend and no effect of treatment could be envisaged. In upper segment, though the trend was similar to the lower one, in α- and β-galactosidase NAA treated segment had more activity. Invertase activity also did not show a clear trend to implicate its function in hypocotyl elongation growth. The results are discussed in relation to establishing a correlation between an activity (glycosidase and invertase) and a physiological process (hypocotyl elongation). It is concluded that these wall-loosening enzymes have no role in elongation growth of Phaseolus vulgaris hypocotyls.     

Polar transport of kinetin in tissues of radish

Polar transport of kinetin-8-¹⁴C occurred in segments of petioles, hypocotyls, and roots of radish (Raphanus sativus L.). The polarity was basipetal in petioles and hypocotyls and acropetal in roots. In segments excised from seedlings with fully expanded cotyledons, indole-3-acetic acid was required for polarity to develop. In hypocotyl segments isolated at this stage, basipetal and acropetal movements were equal during the first 12 hours of auxin treatment after which time acropetal movement declined. Pretreatment with auxin eliminated this delay in the appearance of polarity. In hypocotyl segments excised from seedlings with expanding cotyledons, exogenous auxin was unnecessary for polarity. Potassium cyanide abolished polarity at both stages of growth by allowing increased acropetal movement. The rate of accumulation of kinetin in receiver blocks was greater than the in vivo increase in cytokinin content of developing radish roots.     

Ribosomes Regulate the Stability and Action of the Exoribonuclease RNase R

Ribonucleases play an important role in RNA metabolism. Yet, they are also potentially destructive enzymes whose activity must be controlled. Here we describe a novel regulatory mechanism affecting RNase R, a 3′ to 5′ exoribonuclease able to act on essentially all RNAs including those with extensive secondary structure. Most RNase R is sequestered on ribosomes in growing cells where it is stable and participates in trans-translation. In contrast, the free form of the enzyme, which is deleterious to cells, is extremely unstable, turning over with a half-life of 2 min. RNase R binding to ribosomes is dependent on transfer-messenger RNA (tmRNA)-SmpB, nonstop mRNA, and the modified form of ribosomal protein S12. Degradation of the free form of RNase R also requires tmRNA-SmpB, but this process is independent of ribosomes, indicating two distinct roles for tmRNA-SmpB. Inhibition of RNase R binding to ribosomes leads to slower growth and a massive increase in RNA degradation. These studies indicate a previously unknown role for ribosomes in cellular homeostasis.     

Regulation of alpha-amylase activity in bean stem tissues

α-Amylase activity was assayed in 1-centimeter sections taken from bean (Phaseolus vulgaris var. Kentucky Wonder) hypocotyls and epicotyls at measured distances from the cotyledons. The activity was low throughout the hypocotyl for the first 7 days. An increase was first observed with etiolated hypocotyls in the basal region, becoming higher in the more central regions by 14 to 17 days. By 21 days the activity was highest in the upper region, but had decreased in the lower regions. A comparable pattern was observed for the epicotyl from etiolated seedlings, the activity increasing first in the region closest to the cotyledons. These increases were associated with loss of cells from the pith in the hypocotyl and epicotyl of both dark- and light-grown plants. Since the changes were observed in tissues virtually devoid of starch, it is hypothesized that the control mechanism is related to the cellular disassembly associated with the mobilization of materials released during senescence rather than to a regulation by the enzyme's substrate or products.