Biosynthesis of the Cyanogenic Glucoside Dhurrin in Seedlings of Sorghum bicolor (L.) Moench and Partial Purification of the Enzyme System Involved

The cyanogenic glucoside dhurrin is rapidly synthesized in etiolated seedlings of Sorghum bicolor (L.) Moench. The dhurrin content of the seedlings increases sigmoidally with the germination time. Shoots of 10 centimeters height contain 850 nanomoles of dhurrin per shoot corresponding to 6% of the dry weight. The biosynthetic activity sharply rises upon germination and reaches a maximum level of 10 nanomoles dhurrin/(hour × shoot) after 48 hours when the shoots are 3 centimeters high. This maximum level is followed by a sharp decline in activity when germination time exceeds 65 hours. Dhurrin and the dhurrin-synthesizing enzyme system are primarily located in the upper part of the etiolated shoot where both are evenly distributed between the coleoptile, the primary leaves and the upper 0.5 centimeter of the first internode including the shoot apex. Dhurrin constitutes 30% of the dry weight of the upper 1.2 centimeter of 10 centimeter high shoots. The seed and root contain neither dhurrin nor the dhurrin-synthesizing enzyme system. The codistribution of dhurrin and the enzyme system throughout the seedling indicates that production and storage sites are located within the same cell. Purification of the dhurrin-synthesizing enzyme by gel filtration or by sucrose gradient centrifugations results in a tenfold increase in specific activity. Further purification is accompained by a decline in specific activity due to loss of essential components as demonstrated by reconstitution experiments.     

Electrotropism of Maize Roots : Role of the Root Cap and Relationship to Gravitropism

We examined the kinetics of electrotropic curvature in solutions of low electrolyte concentration using primary roots of maize (Zea mays L., variety Merit). When submerged in oxygenated solution across which an electric field was applied, the roots curved rapidly and strongly toward the positive electrode (anode). The strength of the electrotropic response increased and the latent period decreased with increasing field strength. At a field strength of 7.5 volts per centimeter the latent period was 6.6 minutes and curvature reached 60 degrees in about 1 hour. For electric fields greater than 10 volts per centimeter the latent period was less than 1 minute. There was no response to electric fields less than 2.8 volts per centimeter. Both electrotropism and growth were inhibited when indoleacetic acid (10 micromolar) was included in the medium. The auxin transport inhibitor pyrenoylbenzoic acid strongly inhibited electrotropism without inhibiting growth. Electrotropism was enhanced by treatments that interfere with gravitropism, e.g. decapping the roots or pretreating them with ethyleneglycol-bis-[β-ethylether]-N,N,N′,N′-tetraacetic acid. Similarly, roots of agravitropic pea (Pisum sativum, variety Ageotropum) seedlings were more responsive to electrotropic stimulation than roots of normal (variety Alaska) seedlings. The data indicate that the early steps of gravitropism and electrotropism occur by independent mechanisms. However, the motor mechanisms of the two responses may have features in common since auxin and auxin transport inhibitors reduced both gravitropism and electrotropism.     

Morphogenesis in selaginella: auxin transport in the stem

Selaginella willdenovii Baker is a prostrate vascular cryptogam with a dorsiventral stem. At each major branching of the stem apex a dorsal and a ventral angle meristem is formed. The ventral meristem becomes determined as a root, and the dorsal meristem as a shoot. The present investigation examined the distribution and transport of ¹⁴C-indoleacetic acid through stem tissues as a basis for the pattern of meristem determination. Externally applied indoleacetic acid is transported into receiver blocks with a velocity of 12 millimeters per hour. Much of the auxin becomes immobilized in the tissue and is not transported. The polar ratio of auxin transport is approximately 2. Auxin is transported equally on the dorsal and the ventral sides of the stem axis, and the auxin flux in vascular tissue is twice that in the cortex. In the branch junctions twice as much auxin is transported on the dorsal side as on the ventral side, and this is held to be the consequence of the lateral branch vascular tissue connecting with the dorsal and median, but not with the ventral vascular strand of the stem axis.     

Cysteine, gamma-Glutamylcysteine, and Glutathione Levels in Maize Seedlings : Distribution and Translocation in Normal and Cadmium-Exposed Plants

The levels of cysteine (Cys), γ-glutamylcysteine (γEC), and glutathione (GSH) were measured in the endosperms, scutella, roots, and shoots of maize (Zea mays L.) seedlings. GSH was the major thiol in roots, shoots, and scutella, Cys predominated in endosperms. The endosperm, scutellum, and functional phloem translocation were required for maintenance of GSH pools in roots and shoots of 6-day-old seedlings. Exposure of roots to 3 micromolar Cd, besides causing a decline in GSH, caused an accumulation of γEC, as if the activity of GSH synthetase was reduced in vivo. [³⁵S]Cys injected into endosperms of seedlings was partly metabolized to [³⁵S]sulfate. The scutella absorbed both [³⁵S]sulfate and [³⁵S]Cys and transformed 68 to 87% of the radioactivity into [³⁵S]GSH. [³⁵S]GSH was translocated to roots and shoots in proportion to the tissue fresh weight. Taken together, the data supported the hypothesis that Cys from the endosperm is absorbed by the scutellum and used to synthesize GSH for transfer through the phloem to the root and shoot. The estimated flux of GSH to the roots was 35 to 60 nanomoles per gram per hour, which totally accounted for the small gain in GSH in roots between days 6 and 7. For Cd-treated roots the GSH influx was similar, yet the GSH pool did not recover to control levels within 24 hours. The estimated flux of GSH to the entire shoot was like that to the roots; however, it was low (11-13 nanomoles per gram per hour) to the first leaf and high (76-135 nanomoles per gram per hour) to the second and younger leaves.     

Transport Gradient of Indoleacetic Acid in Pine Seedlings

Movement of indoleacetic acid (IAA-1-¹⁴C) through 5-min segments from stems and roots of Pinus taeda L., P. jeffreyi Gtrev. and Balf., and P. ponderosa Laws, seedlings was measured. All three species showed the same general pattern of IAA-1-¹⁴C transport. Predominantly downward movement occurred at all points along the stem and root; least IAA-1-¹⁴C transport occurred in segments obtained near the shoot apex; and most occurred in segments obtained near or just below the cotyledons. This pattern of transport gradient is different from that reported in other plants. These results could be interpreted in either of two ways: (1) that transport limitations may control movement of the growth-regulator, or (2) that the transport gradient may be an indication of endogenous growth-regulator levels.     

Willow regrowth after galling increases bud production through increased shoot survival

Insect herbivory can negatively or positively affect plant performance. We examined how a stem gall midge Rabdophaga rigidae affects the survival, growth, and bud production of current year shoots of the willow Salix eriocarpa. In mid-May, the gall midge initiates stem galls on the apical regions of shoots. The following spring, galled shoots had thicker basal diameters and more lateral shoots than ungalled shoots. Although galled shoots were on average 1.6 times longer than ungalled shoots, there were no significant differences in shoot length or in the numbers of reproductive, vegetative, and dormant buds per shoot. However, the subsequent survival of galled shoots was significantly higher than that of ungalled shoots, probably because of the thicker basal diameter. This increased shoot survival resulted in approximately two times greater reproductive, vegetative, and dormant bud production on galled shoots compared with ungalled shoots in the following spring. These results suggest that the willow regrowth induced by galling can lead to an increase in bud production through increased shoot survival.     

Physiology of Pinus strobus L. Seedlings Grown under High or Low Soil Moisture Conditions

White pine seedlings (Pinus strobus L.) were grown under highor low soil-moisture levels. The increase in the length andin the fresh weight of seedlings, respiration, photosynthesis,transpiration, translocation of photosynthate from shoots toroots, and bio-electric potentials between the tip and the baseof a stem were measured throughout the growing season from Aprilto October 1964. At both moisture levels the lowest translocation of recent photosynthatefrom shoots to roots occurred during early summer, or at thetime when the rate of root growth was the lowest and that ofthe shoot the highest. The specific activity of ¹⁴CO₂, respiredby the shoots of such plants remained high throughout the 8h of the experiment, indicating a continuous utilization ofrecent photosynthate as a respiratory substrate. On the otherhand, early and late in the growing season, when translocationof recent photosynthate from shoots to roots and the rate ofroot growth were high, the specific activity of ¹⁴CO₂, respiredby the shoots rapidly decreased during the 8 h of the experiment,indicating a drop in the utilization of recent photosynthateas respiratory substrate. The highest positive values for thepotential difference between the top and the base of the mainshoot also occurred in early summer or during the period ofhigh rates of transpiration per needle stomatal surface area.     

Changes in k, rb, and na transport to shoots after anoxia

The effect of anoxia on subsequent uptake and transport of K, Rb, and Na was examined with seedlings of barley (Hordeum vulgare L.), corn (Zea mays L.), and tall fescue (Lolium × Festuca hybrid derivative) to further our understanding of xylem loading. Roots were incubated in solutions depleted of O₂ by flushing with N₂ gas. After 1 hour exposure, plants were returned to aerated solutions for 16 hours prior to measuring uptake and transport. For each species, anoxia pretreatment significantly enhanced Na transport to the shoot. The rate of Na accumulation into roots, however, was not affected. There was no enhancement of either K or Rb accumulation in shoots, indicating specificity for Na transport. A minimum exposure to anoxia of 30 minutes and a minimum of 12 hours elapsed time was necessary to achieve the maximum rate of Na transport to the shoot in barley seedlings. Accumulation of Na in the shoot of both the control and anoxia pretreated barley plants was inhibited by anoxia and by addition of the proline analog, l-azetidine-2-carboxylic acid, during the uptake period. Enhancement of Na transport was associated with a proportional increase in the rate of synthesis of a membrane bound protein with a molecular weight of 78,000 daltons.     

Effects of red, far red, and blue light on enhancement of nitrate reductase activity and on nitrate uptake in etiolated rice seedlings

The effects of red (R), far red (FR), or blue light (B) on the enhancement of nitrate reductase (NR) activity and on nitrate uptake in etiolated rice seedlings were examined. On 5-minute illumination followed by 12-hour dark, R caused marked increase of NR activity, but FR and B caused only slight increase. Illumination with 560 ergs per square centimeter per second of R for 5 minutes caused maximal increase. The effect of R was almost completely counteracted by subsequent illumination with 2,000 ergs per square centimeter per second of FR for 10 minutes, indicating that NR induction was mediated by phytochrome. Exogenous supply of inducer nitrate was not required during the 5-minute illumination and the R-FR cycles, if the seedlings were transferred to nitrate solution at the beginning of the dark incubation. NR activity in the shoots was found high when shoots were illuminated but was low when only roots were illuminated. On continuous illumination for 12 hours, B had more effect on NR increase than R.     

Effects of shoot bending of apple trees on accumulation and translocation of14C-labelled assimilates

The translocation of¹⁴C-labelled assimilates from a single leaf in bent and intact apple shoots was studied in varying stages of shoot development. In actively growing shoots¹⁴C-labelled assimilates translocated from the treated leaf and accumulated mainly in the shoot apex. In moderately growing apple shoots radioactive assimilates were translocated from the treated leaf in both directions towards and down the shoot. In apple shoots showing only slight growth activity the¹⁴C-labelled assimilates were transported from the treated leaf mainly to the base of the shoot, stem and roots. Bending shoots changed the pattern of distribution of radioactive assimilates. Bending actively and moderately growing shoots resulted in higher concentration of 14-carbon in the shoot apex than in controls. In slowly growing and non-growing apple shoots bending caused a higher accumulation of radioactive assimilates in the bent section than in an equal section of control shoots.     

Ethylene Evolution from Maize (Zea mays L.) Seedling Roots and Shoots in Response to Mechanical Impedance

The effect of mechanical impedance on ethylene evolution and growth of preemergent maize (Zea mays L.) seedlings was investigated by pressurizing the growth medium in triaxial cells in a controlled environment. Pressure increased the bulk density of the medium and thus the resistance to growth. The elongation of maize primary roots and preemergent shoots was severely hindered by applied pressures as low as 10 kilopascals. Following a steep decline in elongation at low pressures, both shoots and roots responded to additional pressure in a linear manner, but shoots were more severely affected than roots at higher pressures. Radial expansion was promoted in both organs by mechanical impedance. Primary roots typically became thinner during the experimental period when grown unimpeded. In contrast, pressures as low as 25 kilopascals caused a 25% increase in root tip diameter. Shoots showed a slight enhancement of radial expansion; however, in contrast to roots, the shoots increased in diameter even when growing unimpeded. Such morphological changes were not evident until at least 3 hours after initiation of treatment. All levels of applied pressure promoted ethylene evolution as early as 1 hour after application of pressure. After 1 hour, ethylene evolution rates had increased 10, 32, 70, and 255% at 25, 50, 75, and 100 kilopascals respectively, and continued to increase linearly for at least 10 hours. When intact corn seedlings were subjected to a series of hourly cycles of pressure, followed by relaxation, ethylene production rates increased or decreased rapidly, illustrating tight coupling between mechanical impedance and tissue response. Seedlings exposed to 1 microliter of ethylene per liter showed symptoms similar to those shown by plants grown under mechanical impedance. Root diameter increased 5 times as much as the shoot diameter. Pretreatment with 10 micromolar aminoethoxyvinyl glycine plus 1 micromolar silver thiosulfate maintained ethylene production rates of impeded seedlings at basal levels and restored shoot and root extension to 84 and 90% of unimpeded values, respectively. Our results support the hypothesis that ethylene plays a pivotal role in the regulation of plant tissue response to mechanical impedance.     

The Influence of Shoots, Roots, and Hormones on Sucrose Distribution

The effects of hormones on sucrose distribution were studiedin relation to the control of the relative growth of the shootand the root. The roots and the shoots above the cotyledonsof bean seedlings were removed and the remaining explants allowedto regenerate so that they consisted of growing buds, a segmentof the hypocotyl, and short growing roots. [¹⁴C]Sucrose wasapplied to the middle of the hypocotyl and its distributionwas measured in relation to the presence of the shoots and rootsand to their replacement by auxin and a cytokinin. It was foundthat radioactivity moved towards both the shoot and the root,and that removing one growing region greatly reduced the transportin its direction and increased the relative transport in theopposite direction. Both hormones restored a large part of themovement of radioactive sucrose when they replaced any one ofthe growing regions. However, there was some preferential actionof benzyladenine when it replaced the buds and of auxin whenit replaced the roots. The results indicate that in the intactplant the development of the shoot and the root is correlatedby a positive feedback involving auxin and cytokinins, and themovement of sucrose is controlled indirectly by the sink activityof these growing regions. This control of the movement of sucrose,and presumably other metabolites, accounts for the short-termcompetitive relations between the shoot and the root.     

Micropropagation of juvenile and adult Sorbus domestica L.

Successful propagation of seedlings and mature trees of Sorbus domestica L. has been achieved by in vitro methods. Multiple shoot formation was obtained by placing shoot apices or nodal segments on a modified Schenck and Hildebrandt medium containing benzyladenine. Regenerated shoots were excised and induced to root on media with auxin. In the best treatments 75–85% of shoots from juvenile material rooted. Rooting capacity of shoots from mature explants was lower (30%) and was not improved by dipping the base of shoots in concentration solutions of indolebutyric or naphthaleneacetic acids. Plantlets were ultimately established in soil.Abbreviations BA benzyladenine - IAA indoleacetic acid - IBA indolebutyric acid - NAA naphthaleneacetic acid     

Density gradient localization of plasma membrane and tonoplast from storage tissue of growing and dormant red beet : characterization of proton-transport and ATPase in tonoplast vesicles

Membranes from homogenates of growing and of dormant storage roots of red beet (Beta vulgaris L.) were centrifuged on linear sucrose gradients. Vanadate-sensitive ATPase activity, a marker for plasma membrane, peaked at 38% to 40% sucrose (1.165-1.175 grams per cubic centimeter) in the case of growing material but moved to as low as 30% sucrose (1.127 grams per cubic centimeter) during dormancy.

A band of nitrate-sensitive ATPase was found at sucrose concentrations of 25% to 28% or less (around 1.10 grams per cubic centimeter) for both growing and dormant material. This band showed proton transport into membrane vesicles, as measured by the quenching of fluorescence of acridine orange in the presence of ATP and Mg²⁺. The vesicles were collected on a 10/23% sucrose step gradient. The phosphate hydrolyzing activity was Mg dependent, relatively substrate specific for ATP (ATP > GTP > UTP > CTP = 0) and increased up to 4-fold by ionophores. The ATPase activity showed a high but variable pH optimum, was stimulated by Cl⁻, but was unaffected by monovalent cations. It was inhibited about 50% by 10 nanomolar mersalyl, 20 micromolar N,N′-dicyclohexylcarbodiimide, 80 micromolar diethylstilbestrol, or 20 millimolar NO₃⁻; but was insensitive to molybdate, vanadate, oligomycin, and azide. Proton transport into vesicles from the 10/23% sucrose interface was stimulated by Cl⁻, inhibited by NO₃⁻, and showed a high pH optimum and a substrate specificity similar to the ATPase, including some proton transport driven by GTP and UTP.

The low density of the vesicles (1.10 grams per cubic centimeter) plus the properties of H⁺ transport and ATPase activity are similar to the reported properties of intact vacuoles of red beet and other materials. We conclude that the low density, H⁺-pumping ATPase of red beets originated from the tonoplast. Tonoplast H⁺-ATPases with similar properties appear to be widely distributed in higher plants and fungi.

     

Correlative inhibition of lateral bud growth in Phaseolus vulgaris L. timing of bud growth following decapitation

Summary On intact, 3-week-old plants of Phaseolus the larger bud in the axils of the primary leaves shows slow, continuous elongation growth. Release from correlative inhibition can be detected within 30 min following decapitation. When 0.1% indoleacetic acid in lanolin is applied to the decapitated stem stump, the lateral bud shows slow growth during the first 7 h, then stops completely for a further 15 h but after 2 days a further gradual increase in length is observed.The movement of ¹⁴C-labelled assimilates from the subtending primary leaf into the lateral bud increases following removal of the shoot apex. When indole acetic acid is applied to decapitated plants the ability of the buds to import ¹⁴C increases for 5–7 h and then declines to a negligible amount. Little or no radioactivity from tritiated indoleacetic acid is transported into the lateral buds of decapitated plants during the first 48 h following removal of the apex and it appears that rapid metabolism of the compound occurs in the stem tissues.     

Transport of [1-14C]-indole-3-acetic acid in Abies balsamea shoots ringed with Ethrel

The terminal (1-year-old) shoot of dormant, 2-year-old Abies balsamea (L.) Mill. seedlings was ringed with 0 or 10 mg Ethrel g^-1 lanolin. After 5 weeks of culture under environmental conditions favorable for growth, some of the treated shoots were harvested to measure tracheid number by microscopy and ethylene evolution by gas chromatography-flame ionization detection. The remaining shoots were used to measure basipetal IAA transport in the cambial region by decapitating the shoot apex, applying a pulse of [1-¹⁴C]-IAA to the cut surface, and monitoring the subsequent distribution of radioactivity. Ringing with 10 mg Ethrel g^-1 lanolin, compared with lanolin alone, stimulated cambial region ethylene evolution about 26-fold at, and 3-fold above and below the ringing site, but promoted tracheid production at the ringing site only. Ethrel ringing also increased the velocity, after 26 h transport, at which the front of the [1-¹⁴C]-IAA pulse moved below the ringing site. After 72 h of [1-¹⁴C]-IAA transport, when only immobilized radioactivity was present, the amount of radioactivity recovered in shoots ringed with 10 mg Ethrel g^-1 lanolin was higher than with lanolin alone at the ringing site but lower below it. No difference was observed above the ringing site. The distribution of radioactivity was the same in shoots ringed with lanolin alone and in unringed shoots. The results support the hypothesis that an abnormally high cambial region concentration of ethylene derived from Ethrel ringing inhibits the capacity of basipetal IAA transport at the ringing site, resulting in a local accumulation of IAA that stimulates tracheid production.     

Maintenance of 32P uptake and transport in Pinus serotina seedlings under hypoxic growth conditions

In the present study, we examined the effects of long- and short-term hypoxia on net uptake and transport of phosphorus to shoots of pond pine (Pinus serotina Michx.), a moderately flood-tolerant southern pine, and the influence aerenchyma formation might have in maintenance of P uptake and transport. Seedlings were grown under aerobic (250 μM O₂) or hypoxic (≤50 μM O₂) solution conditions for 5.3 weeks in continuously flowing solution culture containing 100 μM P. Intact seedlings were then labeled with ³²P for up to 24 h to determine how short- and long-term hypoxic solution conditions affected rates of unidirectional influx and the accumulation of ³²P in roots and shoots. Seedlings in the long-term hypoxic treatment were grown for 5.3 weeks in hypoxic solution and also labeled in hypoxic uptake solution. The short-term hypoxic treatments included a 24-h hypoxic pretreatment followed by time in labeled hypoxic uptake solution for seedlings grown under aerobic or hypoxic conditions; in the latter case, diffusion of atmospheric O₂ entry into stem and root collar lenticels was blocked, thus removing any influence that aerenchyma formation might have had on enhancing O₂ concentrations of root tissue. Although unidirectional influx rates of ³²P in roots of seedlings grown under long-term hypoxic conditions were 1.4 times those of aerobically grown seedlings, accumulation of ³²P in roots was similar after 24 h in labeled uptake solution. These results suggest that ³²P efflux was also higher under hypoxic conditions. Higher shoot/root fresh weight ratios and lower shoot P concentrations in seedlings grown under hypoxic solution conditions suggest that the “shoot P demand” per unit root should be high. Yet accumulation of ³²P in shoots was reduced by 50% after 24 h in hypoxic uptake solution. Both short-term hypoxic treatments decreased accumulation of ³²P in roots by more than 50%. Short-term hypoxia decreased shoot accumulation in seedlings grown under aerobic and hypoxic conditions by 84 and 50%. respectively. Short- and long-term hypoxic conditions increased the percentage of root ³²P in the nucleic acid and chelated-P pools, resulting in a significantly smaller percentage of ³²P in the soluble inorganic phosphate (p_i) pool, the pool available for transport to the shoot. However, a reduction in pool size or in labeling of the pool available for transport cannot fully account for the large reduction in accumulation of ³²P in shoots, particularly in the short-term hypoxic treatment of aerobically grown seedlings. Our results suggest that both influx and transport of ³²P to shoots of pond pine seedlings are O₂-dependent processes, and that the transport of ³²P to shoots may be more sensitive to hypoxic solution conditions than influx at the cortical and epidermal plasmalemma, with aerenchyma formation supporting a substantial amount of both ³²P uptake and transport.     

In vitro regeneration of plantlets in Brassica alboglabra

Different vegetative parts of Brassica alboglabra seedlings and mature plants were used as explants in culture.A high frequency (60–100%) of shoot regeneration was obtained from hypocotyl explants, nodal stem segments, internodal segments and shoot apices cultured on Murashige-Skoog basal medium. Addition of 6-benzylaminopurine and kinetin increased the average number of shoots per explant. When detached and transferred to basal medium, the shoots readily developed roots. Regenerated plantlets could be successfully transplanted in soil.     

Rhythmic Differences in the Basipetal Movement of Indoleacetic Acid between Separated Upper and Lower Halves of Geotropically Stimulated Corn Coleoptiles

Rhythmic fluctuation in the basipetal movement of auxin occurs in corn (Zea mays) coleoptiles oriented either in the vertical or in the horizontal position. This periodicity of transport rate varies from region to region in a horizontal coleoptile. Between an upper and lower half coleoptile (with respect to gravity), the comparable regions in the coleoptile do not exhibit similar periods. The velocity of transport also varies from region to region along a geostimulated coleoptile. In the upper half coleoptile, the velocities are 29 millimeters per hour (tip), 8 millimeters per hour (mid), and 30 millimeters per hour (base); in the lower, 41 millimeters per hour (tip), 12 millimeters per hour (mid) and 12 millimeters per hour (base).     

The distribution of diosgenin in Dioscorea spp

The distribution of diosgenin in Dioscorea deltoidea Wallich and D. sylvatica Ecklon at successive stages of growth is described. Diosgenin occurs in the cotyledon-endosperm of the dormant seeds, and in the tubers, roots, stems and leaves of plants from the seedling stage onwards. It accumulates in the tubers of seedlings grown in daylight, but not in those of seedlings grown in darkness. Actively growing aerial tissues such as leader shoots appear to be the sites of formation from which the diosgenin is translocated to the tubers. The diosgenin is evenly distributed throughout the tubers.