14C fixation,metabolic labeling patterns,and translocation profiles during leaf development in Populus deltoides

The incorporation of photosynthetically fixed ¹⁴CO₂ and the distribution of ¹⁴C among the main chemical constituents of laminae and petioles were examined in cottonwood (Populus deltoides Bartr. ex Marsh.) leaves ranging in age from Leaf Plastochron Index (LPI) 3 (about one-quarter to one-third expanded) to LPI 30 (beginning of senescence). In addition, carbon flow among chemical fractions and translocation from leaves of LPI 7 and 14 were examined periodically up to 24 h after labeling. Specific activity of ¹⁴C (on dry-weight basis) increased in developing laminae to full leaf expansion, decreased in the mature leaves to LPI 16, then remained constant to LPI 30. In developing leaves (LPI 3-5), after 2 h, most of the ¹⁴C was found in protein, pigments, lipids, and other structural and metabolic components necessary for cell development; only 28% was in the sugar fraction of the lamina. In fully expanded leaves (LPI 6-8), after 2 h, the sugar fraction contained 50–60% and about 90% of fixed ¹⁴C in the lamina and the petiole, respectively. In a pulsechase kinetic series with recently mature leaves, 60% of the ¹⁴C was found in the sugar fraction after 15 min of ¹⁴CO₂ fixation. Over the 24-h translocation period, ¹⁴C decreased in sugars to 23% and increased in the combined residue fraction (protein, starch, and structural carbohydrates) to about 60% of the total activity left in the lamina. Within 24 h after labeling, the turnover of ¹⁴C-organic acids,-sugar, and-amino acids (either metabolzed or translocated from the leaf) was 30, 70 and 80%, respectively, of that initially incorporated into these fractions by a leaf at LPI 7 (turnover was 55% of ¹⁴C-organic acids, 80% of ¹⁴C-sugar, and 95% of ¹⁴C-amino acids at LPI 14). Anatomical maturity in cottonwood leaves is closely correlated with physiological maturity and with production of translocatable sugar.Abbreviations LPI leaf plastochron index - PI plastochron index Research Plant Physiologist and Chief Plant Physiologist, respectively     

Shoot regeneration from petioles of coral bells (Heuchera sanguinea Engelm.) cultured in vitro, and subsequent planting and flowering ex vitro

Summary Successful shoot regeneration from petioles, leaves, and petioles with leaves cultured in vitro is reported in Heuchera sanguinea. Petioles or petioles with leaves regenerated more shoots than leaves alone. For culture, the optimum hormonal concentrations were 0.19 μM α-naphthaleneacetic acid combined with 0.44 or 4.4 μM benzyladenine in Murashige and Skoog-based (MS) medium: the regenerating rate and the number of shoots per explant were 60% and 8.6–9.7, respectively. Histological study on petiole culture showed dividing cell clusters including vascular tissues after 1 wk, callus including several dividing cell clusters at the periphery after 3 wk and then apical meristems with immature leaves after 5 wk. Rooting from the regenerated shoots was highest (95%) on MS medium containing 4.9 μM indole-3-butyric acid. Seventy-three percent of rooted plants were successfully acclimatized in pots. When they were cultured in the field, the plants grew and most flowered the following year over winter.     

APOLAR MOVEMENT OF ZEATIN THROUGH COLEUS PETIOLES AND PISUM ROOTS AS ESTIMATED BY BIOASSAY AND RADIOACTIVE LABELLING

The movement and polarity of zeatin, a highly active, endogenous cytokinin, through petioles and roots were tested in the classical experimental arrangement using excised 5-mm sections. Zeatin in the receiver cylinders of agar was measured by soybean callus bioassay and by liquid scintillation counting of ¹⁴C that had been added in the donor cylinders as [8-¹⁴C] zeatin. Both methods agreed in showing movement, but there was no polarity in Coleus #5 petioles. The amounts moved were about one-tenth of the GA-3 movement through petioles of the third pair of leaves of the same clone. Movement of ¹⁴C-zeatin through Pisum roots was similarly statistically significant but non-polar; the amounts moved were similar to those previously observed for polar GA-3 movement through Zea roots.     

The Effects of Benzylaminopurine on Growth and 14Carbon Translocation in Excised Mustard Cotyledons

When localized areas of blades of recently excised mustard cotyledons were fed with ¹⁴CO₂ it was found that the fixation products passed rapidly into the veins and then were translocated to the petiole. Since other cotyledons similarly treated subsequently rooted at the petiole base, this suggested that it acted as a sink for assimilates. Treatment of excised cotyledons with benzylaminopurine (BAP) resulted in the enhancement of blade growth and the suppression of root initiation and development. Quantitative determinations of labelled assimilate in both the cotyledon blade and the terminal 2 mm portion of the petiole were made. It was found that cotyledons treated with BAP at a concentration high enough to suppress completely root initiation had a higher level of radioactivity in the petiole base than the terminal segments in untreated petioles, whether expressed as specific activity or as a percentage of the total radiocarbon fixed. BAP-treated cotyledons also fixed consistently higher levels of ¹⁴CO₂, probably due to an increase in photosynthetic area. The results suggest that BAP alters the pattern of differentiation at the base of the petiole rather than the polarity of movement of assimilates to the petiole base.     

Failure of Ethylene to Change the Distribution of Indoleacetic Acid in the Petiole of Coleus blumei X frederici during Epinasty

The effect of ethylene on the distribution of applied indoleacetic acid in the petiole of Coleus blumei Benth. X C. frederici G. Taylor has been investigated during the development of epinastic curvature. Using intact plants, ¹⁴C-IAA was applied to the distal region of the leaf lamina and the accumulation of label in the abaxial and adaxial halves of 5 mm petiole sections was determined after 1.5, 3, and 6 hours. Over this period the label was transported out of the lamina into the petiole at a rate of at least 66 mm hr⁻¹. Of the total amount of label in the petiole sections, 24 to 30% was located in the adaxial half and this distribution was not altered significantly by exposing plants to an atmosphere containing 50 μl/l ethylene. Thus when epinastic curvature is induced by ethylene there is no associated increase in the IAA content of the expanding adaxial half. The role of endogenous IAA in petiole epinasty was studied by restricting its movement with DPX 1840 (3,3a-dihydro-2-[p-methoxyphenyl]-8H-pyrozolo{5,1-a}isoindol-8-one). The leaf petioles still showed an initial epinastic response to ethylene. It is concluded that ethylene-induced epinasty is not dependent upon either any change in the transport of IAA or its redistribution within the petiole.     

14C UPTAKE BY THE MARINE ANGIOSPERM PHYLLOSPADIX SCOULERP

Rhizomes and attached leaves of Phyllospadix scouleri Hook, were collected in the intertidal zone along the central California coast and exposed to a solution of NaH¹⁴CO₃ in seawater under controlled laboratory conditions. Over a 90-min period roots and rhizomes absorbed very little ¹⁴C compared to leaves. Translocation during that time was minor. Plants pretreated with the photosynthetic inhibitor DCMU showed no ¹⁴C uptake, indicating that under normal circumstances the carbon which is absorbed by leaves is fixed and accumulates as photosynthate. The rate of gross photosynthesis was about 13 mg CO₂ g dry wt⁻¹ hr⁻¹. Gross photosynthesis of wet leaves exposed to ¹⁴CO₂ in air was significantly less than leaves exposed to NaH¹⁴CO₃. The effect of a leaf-grazing limpet (Notoacmea paleacea) on leaf anatomy and ¹⁴C uptake is discussed.     

In Vivo Measurement of Indole-3-acetic Acid Decarboxylation in Aging Coleus Petiole Sections

The concentration of indoleacetic acid (IAA) in plant tissues is regulated, in part, by its rate of decarboxylation. However, the commonly used in vitro assays for IAA oxidase may not accurately reflect total in vivo decarboxylation rates. A method for measuring in vivo decarboxylation was utilized in which ¹⁴CO₂ is collected following uptake of [1-¹⁴C]IAA by excised tissue sections. After a 30-minute equilibration period, the evolution of ¹⁴CO₂ was found to follow an approximately linear course with respect to both time and tissue weight.

Decarboxylation rates were measured by this method in petiole sections of the Princeton clone of Coleus blumei Benth. Both the ¹⁴CO₂ evolved per milligram tissue and the percent of [1-¹⁴C]IAA uptake decarboxylated were highest in sections from the youngest petioles tested, and declined in the older tissue. Thin layer chromatography of acetonitrile extracts from the [1-¹⁴C]IAA-treated petioles showed a decreasing amount of free IAA and an increase at the retardation factor of indoleacetylaspartate in the older sections. The decreased decarboxylation rates in the older petioles may be attributable to a generally lower metabolic rate and increased protection of the IAA by conjugation.

     

Changes in soluble carbohydrates during phytochrome-regulated petiole elongation in watermelon seedlings

Changes in soluble carbohydrate composition and concentration in leavesand petioles of watermelon (Citrullus lanatus (Thunb)Matsum and Nakai cv. Sugar Baby) seedlings during early stages ofphytochrome-regulated petiole elongation were investigated. Watermelon seedlingswere grown in a controlled environment with 350 molm^–2 s^–1 photosynthetically activeradiation (PAR) during a 12-h photoperiod. Low intensity end-of-day(EOD) light treatments (for 15 min) of red (R), far-red (FR) and FRfollowed by R (FR/R) were initiated when the seedlings were 14 days old.Seedling growth, and soluble carbohydrate concentration and composition inleaves and petioles were determined after 3 and 6 days of EOD light treatments.The EOD FR increased the petiole length and dry mass partitioned to petioles asearly as 3 days into the treatment. This increased petiole dry mass inFR-treated plants was accompanied with an increase in reducing sugar (glucoseand fructose) concentration in the petioles. Although both leaves and petiolesshowed this effect, the relative increase was greater in petioles than leaves.While the most abundant sugars in petioles were fructose and glucose, thepredominant sugars in leaves were sucrose, raffinose, and stachyose. Thephotoreversion of FR induced changes in growth and sugar concentrations by Rindicates the involvement of phytochrome in these processes.     

Translocation and incorporation of 14C into the petiole from different regions within developing cottonwood leaves

Summary The ability of a developing cottonwood (Populus deltoides Bartr.) leaf to export ¹⁴C-labeled assimilates begins at the lamina tip and progresses basipetally with increasing LPI. This progression indicates that portions of leaves function quasi-independently in their ability to export ¹⁴C-photosynthate. Although most of the exported radioactivity was recovered in the petiole as water-80% alcohol-soluble compounds, there was also substantial incorporation into the chloroform and insoluble fractions. This observation indicates that assimilates translocated from the lamina are used in structural development of the petiole. Freeze substitution and epoxy embedding were used to prepare microautoradiographs for localization of water-soluble compounds. Radioactivity was found in all cell types within specific subsidiary bundles of the petiole. However, radioactive assimilates appeared to move from the translocation pathway in the phloem toward active sinks in the walls of the expanding metaxylem cells. Translocation in the mature xylem vessels was not observed.     

Uptake, Translocation, and Metabolism of IAA in the Olive (Olea europea): II. THE FATE OF EXOGENOUSLY APPLIED [2-14C]IAA TO DETACHED 'MANZANILLA' OLIVE LEAVES

The uptake, translocation, and metabolic pathway of IAA degradationin detached ‘Manzanilla’ olive leaves were studiedusing [2–¹⁴C] IAA. The uptake of radioactivity was veryrapid. With scraped leaves devoid of their peltate scales, itreached 80 percent of total applied radioactivity after 6 h.Translocation from the site of application in mature leaveswas negligible. Limited transport was apparent only in youngleaves. The absorbed label was initially ethanol-extractablebut later half of the radioactivity could not be extracted inethanol and had to be extracted with 1 N KOH. The first andmajor metabolite of the IAA in the olive leaves was identifiedas indolecarboxylic acid (ICA). Minor amounts of indoleacetylasparticacid (IAsp) and indolealdehyde (IAld) were also detected. Theidentity of the KOH-extractable label was not determined.     

The influence of root temperature on 14C assimilate profiles in wheat roots

Summary The rate of translocation of ¹⁴C assimilates from leaves to seminal roots in wheat seedlings was considerably reduced by lowering root temperature from 20° to 10° or 5° although the total translocation of ¹⁴C to the roots after 24 h was little affected by temperature. The lowered root temperatures (particularly 5°) resulted in a more uniform distribution of assimilate along the roots than did a temperature of 20°, the ratios of radioactivity/cm in the apical cm, elongating zone, and basal parts of the root after 24 h being 14.0:9.6:1 in 20° roots by contrast with 2.8:1:1 in 5° roots. Temperature effects on assimilate distribution may help explain the observations that for roots grown below 15° ion uptake is sustained in older parts and that roots grown at a low temperature are thicker than roots grown at a higher one.     

TRANSLOCATION OF 14C METABOLITES IN CARROT ROOT

Four-month-old carrot plants exposed to ¹⁴CO₂ for 1 hr in light were harvested successively at 1, 5, 10, 26, and 78 hr after initial exposure. Half-mm thick transverse slices from 3 and 10 cm below the crown were frozen quickly, freeze-dried, and autoradiographed on film. Radioactivity was first localized in a ring surrounding the cambium. The radioactive region extended centrifugally along radii as discrete loci to half the phloem thickness in 10 hr, in a pattern similar to radial rows of callose-stained sieve elements. Median longitudinal sections stained for callose demonstrated the presence of anastomosing sieve tube strands between the more vertical sieve tubes of differing age. Radioactive materials did not move across the cambium for 5–10 hr. These data fit with the decreased growth, in earlier studies, of uniform phloem explants removed from increasing distances from the cambium and of the lesser growth than phloem of similar xylem explants.     

THE TIME-COURSE OF POLAR MOVEMENT OF GIBBERELLIN THROUGH ZEA ROOTS

The polarity of movement of gibberellin through sections cut from near the root tips of Zea mays L. was studied, using methods like those we previously used in roots for auxin and in petioles for auxins, cytokinins, and gibberellic acid (GA-3). One μg GA-3 was added in a donor agar block and gibberellin activity in the receiver agar at the opposite end of the section was measured directly with a modified barley endosperm bioassay. The movement of gibberellin was away from the root tip (basipetal) and thus opposite in direction to the polarity of auxin through such root sections. The time-course of basipetal movement was dissimilar to that for gibberellin or auxin movement through petiole sections. It took 14-18 hr for gibberellin activity equivalent to 6 ng GA-3 to collect in the basal receivers on roots. Apical receivers showed activity equivalent to 1.6 ng GA-3 at 14-18 hr. Less than 0.01 ng equivalent GA-3 was collected from sections to which GA-3 was not added, so the 6 and 1.6 ng were almost entirely due to the added GA-3. These general conclusions were confirmed with an experiment using ¹⁴C-GA-3. A decline in activity in receivers was found in some experiments at 18 hr, paralleling earlier results with GA-3, IAA, and adenine in petioles and IAA in roots.     

6(5)CARBOXYFLUORESCEIN AS A TRACER OF PHLOEM SAP TRANSLOCATION

6(5)carboxyfluorescein (6(5)CF), a polar fluorescein with an apparent pK of 6.3, was introduced, as a pH 6.3 solution, into the apoplast of lamina or petioles of mature soybean leaves. Freehand sections were prepared at various times and immediately observed with a fluorescence microscope. 6(5)CF-associated fluorescence appeared in all sink organs, from shoot apex to roots. It was strictly confined to the phloem regions, even after 4 days. Its transport into young leaves ceased at approximately the time they underwent sink-to-source transition. It was never transported between two leaflets of the same leaf. Its transport was interrupted by phloem destruction. All these transport characteristics were highly reproducible, and were paralleled by those of ¹⁴C transport after application of (¹⁴C)sucrose to leaf surfaces. In contrast with 6(5)CF, fluorescein was transported between mature leaves, and between leaflets of the same leaf. It was not restricted to phloem, and often appeared in the xylem region. These results indicate that 6(5)CF can be used to monitor phloem sap translocation in real time, in short- and long-term experiments.     

Movement of Kinetin and Gibberellic Acid in Leaf Petioles during Water Stress-induced Abscission in Cotton

Movement of [¹⁴C]kinetin and [¹⁴C]gibberellic acid was examined in cotton (Gossypium hirsutum L.) cotyledonary petiole sections independent of label uptake or exit from the tissue. Sections 20 millimeters in length were taken from well watered, stressed, and poststressed plants. Transport capacity was determined using a pulse-chase technique. Movement of both kinetin and gibberellic acid was found to be nonpolar with a velocity of 1 millimeter per hour or less, suggesting passive diffusion. Neither water stress nor anaerobic conditions during transport of labeled material affected the transport capacity of the petioles.     

Ethylene and growth control in the fringed waterlily (Nymphoides peltata): Stimulation of cell division and interaction with buoyant tension in petioles

The effect of ethylene on petiole growth of the Fringed Waterlily (Nymphoides peltata (S.G. Gmelin) O. Kuntze) changes during leaf ontogeny. During early development (before expansion of laminae), ethylene causes an increase in both cell number and cell size; later in development, promotion of rapid cell expansion is the dominant effect. The early effects may contribute to the accommodation of new leaves to water columns of different depth. The later effects on cell expansion only are shown to contribute to the rapid accommodation of floating leaves when changes in water level submerge the laminae. This kind of accommodation results from an interaction between accumulated ethylene, which increases wall extensibility, and the tension in petioles due to natural buoyancy which, it is suggested, supplements the driving force for cell expansion. Cell age (position) within a petiole and age of the whole petiole influence the growth response to ethylene alone and the amount of extra growth produced by applying tension when ethylene is present. In young petioles, apical cells are highly sensitive to ethylene and tension causes little further growth; older cells in both immature and mature petioles show little response to ethylene unless the petiole is under tension. Young (but not mature) petioles respond slowly to applied tension even in the absence of ethylene. It is concluded that as cells age the driving force for expansion limits increasingly their capacity to respond to the wall-loosening effects of ethylene. Dual sensitivity to ethylene and buoyant tension facilitates rapid accommodation responses but sensitivity of young petioles to tension alone may exclude Nymphoides from habitats where current velocity is appreciable.     

Apetiolar photosynthate translocation

Apetiolar transport of photosynthate ⁻¹⁴C has been studied by feeding of ¹⁴CO₂ to soybean petioles. Translocation occurs in the absence of leaves, but both the rate and velocity are diminished. The effect of root excision is not as profound as that of leaves. It appears, in some instances, to inhibit transport partially, so that accumulation of photosynthate develops, giving a steeper isotopic gradient. The effect of leaf darkening is to diminish its uptake of photosynthate from the petiole, possibly as a result of decreased transpiration in the lowered temperature of the darkened leaf. The data suggest that neither mass flow nor active transport provide an adequate basis for normal photosynthate transport but that the leaves provide a direct force requiring structural continuity, or a translocation carrier.     

Conductance of needle and twig axis phloem of damaged and intact Norway spruce (Picea abies (L.) Karst.) as investigated by application of14C in situ

Summary Phloem conductance of¹⁴C-labelled assimilates was investigated in natural stands of Norway spruce showing substantial damage from needle yellowing and needle loss disease. Terminal current-year shoots of a branch were allowed to fix¹⁴CO₂ (300–600 ppm in air) and carbon dioxide net uptake was monitored with a gas analyser. The difference between¹⁴C-uptake and the amount of radiocarbon determined in the photosynthesizing needles was interpreted to reflect assimilate export from the needles to the axis of the tree. Compared with an undamaged control tree,¹⁴C-export from the assimilating needles was not impaired in the yellowing tree and only slightly reduced in the tree showing needle loss. Incorporation of¹⁴C into starch increased significantly during autumn particularly in the tree showing needle loss. Import of radiocarbon from the¹⁴C-labelled phloem sap in twig axes and needles older than 1 year was used as a measure of phloem conductivity of older sections of a branch which showed considerable damage. Carbon uptake by these older plant parts was more pronounced than in undamaged twigs. In the case of older needles enhancement of¹⁴C-incorporation suggested an increased sink strength, while the same phenomenon in the twig axes was interpreted as a consequence of partially impaired conductivity of individual sieve elements resulting in an inhomogeneous velocity of phloem transport. The hypothesis is put forward that curtailed viability of the sieve cells is responsible for a delay of transport, which is compensated for by an augmented production of phloem elements from the cambium.     

Changes in the Distribution of 14C-Labelled Assimilates in Sugar-beet with Variation of Temperature

Plants were allowed to assimilate ¹⁴CO₂ for 30 min at 5, 15,25, and 35 °C. The changes in ¹⁴C content of a mature expandedleaf (Leaf 4), young apical leaves, and storage root, were sequentiallyfollowed over a subsequent period of 24 h in continuous light.In a second experiment plants were transferred after ¹⁴CO₂ assimilationto temperatures of 10, 18, 26, and 34 °C, and the partitionof ¹⁴C between the ethanol-soluble and ethanol-insoluble fractionsof the roots and leaves was followed over a period of 72 h. The specific activities of the apical leaves and of the storageroot increased to a maximum 2 h after labelling at 25 °C,4 h at 15 and 35 °C, and 6 h at 5 °C suggesting thatthe optimum temperature for translocation of photosynthate wasabout 25 °C. The ¹⁴C partition to ethanol-soluble and ethanol-insoluble fractionsof the roots and leaves was largely attained in. 9 h. Littlerepartition of ¹⁴C assimilate fractions occurred as a resultof temperature change or growth. Root ethanol-insoluble activity,however, did increase significantly over the 72-h period : possiblecauses of this slow incorporation and their relevance to themechanism of sugar storage are discussed.     

Transport 14C-markierter Assimilate im Phloem von Pelargonium zonale und Phaseolus vulgaris

Zusammenfassung 6–10 min nach Beginn der ¹⁴CO₂-Assimilation befindet sich ¹⁴C-Aktivität im Stiel des ¹⁴CO₂-exponierten Blattes belichteter Pflanzen von Pelargonium zonale und Phaseolus vulgaris. Die Verteilungsrichtung der Assimilate im Sproß scheint vor allem durch das Alter des ¹⁴C-assimilierenden Blattes bestimmt, jedoch nicht einseitig festgelegt zu sein; geringe, aber faßbare Mengen an ¹⁴C-Saccharose wereden innerhalb von 20 min (bei Phaseolus) und 180 min (bei Pelargonium) auch aus relativ jüngeren in ältere Blätter transportiert.Neben ¹⁴C-Saccharose wurden im Blattstiel-und Stengelgewebe stets markierte Zuckerphosphate, Hexosen und organische Säuren nachgewiesen. Stärke war nur zu einem verschwindend geringen Anteil an der Gesamtmarkierung der analysierten Transportstrecken beteiligt.

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Translocation of ¹⁴C-labelled assimilates in the phloem of Pelargonium zonale and Phaseolus vulgaris

Summary After the laminae of leaves of intact plants had been exposed to ¹⁴CO₂ the translocation of ¹⁴C-labelled assimilates across the petioles starts very quickly: 6 to 10 min later ¹⁴C-activity could be detected in the basal part of the petioles. The way of distribution within the plant seems to be influenced mainly by the age of the ¹⁴CO₂-assimilating leaf, however, but not in the sense of an unidirectional movement; little but distinct amounts of ¹⁴C were carried also from younger yet full expanded leaves down to older leaves, within 20 min (in Phaseolus) or 180 min (in Pelargonium). Besides sucrose, which was shown by paper chromatography to be the main form of assimilates translocated in these species, we identified sugar phosphates, hexoses, and some organic acids in the petiole and stem tissue as being radioactive also. In our experiments, the petiole segments did not contain any remarkable amount of ¹⁴C-labelled starch.