Studies on the Movement of Solutes between the Sieve Tubes and Surrounding Tissues3: I. INTERFERENCE BETWEEN SOLUTES AND RATE OF TRANSLOCATION MEASUREMENTS

Certain aspects of the secretion of solutes into, and removalfrom, the sieve tubes of isolated stem segments and rooted cuttingsof Salix viminalis have been studied. Sieve-tube sap was obtainedeither as honeydew from whole individuals or via the severedstylets of the aphid Tuberolachnus salignus (Gmelin). It was shown that interference occurred between the chemicallyunrelated solutes, sucrose and the cations potassium and rubidium.On raising the potassium concentration in the sieve-tube sapby passing a solution of this ion through the xylem, the sucroseconcentration declined. When the sucrose concentration fellover a period of days due to respiratory loss of carbohydratesfrom an isolated stem segment, a concomitant rise in eitherthe potassium or rubidium level in the sap occurred. When a solution of sodium was passed through the xylem, theconcentration of this ion in the sieve-tube sap rose, whilstthat of potassium fell at first, but later rose higher thanits initial value, indicating that both antagonism and synergycan occur between these ions. On introducing both these cationsinto the xylem simultaneously, more sodium than potassium wastaken up by the segment, though the increase in the sodium concentrationin the sieve-tube sap was less than that of the potassium. Perfusingthe xylem with a calcium solution had no effect upon the concentrationof potassium in the sieve tube. It has been shown that the rate of translocation of a solutealong the sieve tube, as measured by the two colony technique,depends upon the rate of removal of this solute from the sievetube. The amount of such lateral loss from the sieve tube isrelated to the potential gradient for a solute between the sievetube and surrounding cells.     

The Movement of Sugars into the Sieve Elements of Bark Strips of Willow: II. EVIDENCE FOR TWO PATHWAYS FROM THE BATHING SOLUTION

It is suggested that when ¹⁴C-labelled sucrose, or its constituenthexoses, are applied to the cambial surface of a bark strip,the sugars can move into the sieve elements by two pathways.The first is a direct one which probably involves the companioncells; in it the labelled sugars do not mix with pools of unlabelledsugars before entry into the sieve elements. Entry by this pathwayleads to activity in a wide range of compounds in the sieve-tubeexudate. The second pathway is an indirect one, involving thestorage parenchyma of the phloem, where considerable metabolicchanges take place involving the sugar applied to the strip,but the products of these changes, apart from sucrose, are unableto move into the sieve elements. These results are discussed in relation to published work onthe cytology of angiosperm phloem, and the results of otherinvestigators on the movement of sugars into plant cells.     

P PROTEIN IN THE PHLOEM OF CUCURBITA : II. The P Protein of Mature Sieve Elements

During maturation of sieve elements in Cucurbita maxima Duchesne, the P-protein bodies (slime bodies) usually disperse in the tonoplast-free cell. In some sieve elements the P-protein bodies fail to disperse. The occurrence of dispersal or nondispersal of P-protein bodies can be related to the position of the sieve elements in the stem or petiole. In the sieve elements within the vascular bundle the bodies normally disperse; in the extrafascicular sieve elements the bodies often fail to disperse. Extrafascicular sieve elements showing partial dispersal also occur. The appearance of the sieve plate in fixed material is related to the degree of dispersal or nondispersal of the P-protein bodies. In sieve elements in which complete dispersal occurs the sieve plate usually has a substantial deposit of callose, and the sieve-plate pores are filled with P protein. In sieve elements containing nondispersing P-protein bodies the sieve plate bears little or no callose, and its pores usually are essentially "open." The dispersed P-protein components may aggregate into loosely organized "strands," which sometimes extend vertically through the cell and continue through the sieve-plate pores; but they may be oriented otherwise in the cell, even transversely.     

Movement of Water and Solutes in Sieve Tubes of Willow in Response to Puncture by Aphid Stylets. Evidence against a mass flow of solution

Experiments are described in which bark strips of willow were sealed to polythene tubes having two compartments. This allowed investigations to be made of the transport along the sieve tubes of tritiated water, ¹⁴C-labelled sugars, and ³²P-phosphates from one compartment, towards a stylet situated in the bark over the other compartment. Although activity from both ¹⁴C and ³²p was detected in the stylet exudate usually within 1 hour from isotope application, tritium activity was never detected even after a period of 8 hours in most experiments, though in certain cases, very low activities were detected after 4 hours. Subsequent experiments in which stylets were sited over both compartments showed that tritium activity moved laterally into the punctured sieve element more rapidly than either ¹⁴C or ³²P. Experiments using both live and dead bark in which stylets were not employed, showed that within 4 hours tritium activity had moved by diffusion along the whole length of a bark strip, therefore after this time tritium activity could have moved into the stylet exudate by a diffusional process. The lack of rapid longitudinal movement of tritiated water along the sieve tubes, indicates that the transport process is unlikely to be a mass flow of solution.     

The Movement of Sugars into the Sieve Elements of Bark Strips of Willow: I. METABOLISM DURING TRANSPORT

¹⁴C-labelled sugars (sucrose, glucose, and fructose) were suppliedto the cambial surface of bark strips of willow, and in conjunctionwith the aphid stylet technique (Weatherley, Peel, and Hill,1959), the movement into and distribution of activity in thesieve tube sap was examined. As well as sucrose, free hexosesand sugar phosphates were found to contain the ¹⁴C-label, andrapid interconversions occurred between these compounds somewherealong the entry route. Sucrose entry into the sieve elementswas accompanied by at least a partial breakdown into its componenthexoses, and the involvement of a sugar phosphate pool was alsodemonstrated. Possible transformation sites on the entry routebetween the solution bathing the cambial surface and the sieveelement vacuole are discussed.     

Studies in the Respiratory and Carbohydrate Metabolism of Plant Tissues1: XVII. THE EFFECT OF IODOACETATE ON THE CO2 OUTPUT, PATHWAYS OF GLUCOSE RESPIRATION AND CONTENTS OF PHOSPHATE ESTERS IN STRAWBERRY LEAVES

When specifically labelled glucose was fed to strawberry leaves,the C₆/C₁, quotient (rate of release of ¹⁴CO₂ from glucose-6-¹⁴C/rateof release of ¹⁴CO₂ from glucose-1¹⁴C ranged from 0.27 to 0.35in leaves in water and from 0.46 to 0.96 in leaves fed withiodoacetate. These quotients indicate that both the glycolyticand the pentose phosphate pathways participate in the respirationof strawberry leaves, with a greater contribution from the formerin the iodoacetate increased CO₂ output. Concurrently with the increase of CO² output in iodoacetate,the contents of glucose-6-phosphate (G6P), fructose-6 (F6P)and fructose-1,6-diphosphate (FDP) increased greatly; therewas a smaller increase of phosphoenol-pyruvate (PEP). The increasein the CO₂ output in iodoacetate may be explained solely onthe basis that the increases of G6P and FDP accelerate the ratesrespectively of the pentose phosphate pathway and of glycolysisand traffic into the tricarboxylic acid cycle. The increasein content of G6P and FDP is attributed to an increase in theaccessibility of enzymes and substrates caused by iodoacetate.Alternatively the increased CO₂ output in iodoacetate may bepartly due to uncoupling of oxidative phosphorylation.     

Comparative Studies of Leaf Tissue and Isolated Mesophyll Protoplasts: II. ION RELATIONS

Freshly isolated tobacco mesophyll protoplasts had contentsof K^$, Cl^– and Na^$ slightly higher (on a cell basis) thanthe original leaf tissue, and had a high K^$/Na^$ ratio similarto that of the leaf tissue. Influxes of these ions into theprotoplasts were of similar magnitudes to the correspondingfluxes in leaf tissue when compared on the basis of the respectiveplasmalemma surface areas. In both systems the K^$ influx wasstrongly inhibited by CN^– and by DNP. These results suggestthat the ion relations of the freshly isolated mesophyll protoplastswere similar to those of the original leaf tissue and that isolatedmesophyll protoplasts should be a useful system for the studyof ion transport processes in leaf cells.     

Studies on Chloroplast Degradation in vivo: III. THE EFFECT OF MAGNESIUM ON ELECTRON TRANSPORT AND PHOSPHORYLATIQN IN PLASTIDS ISOLATED FROM YOUNG AND AGEING COTYLEDONS

The magnesium dependence of NADP-reduction, oxygen evolutionand concomitant ATP-formation was studied as a function of theage of the Cucurbita cotyledons from which the chloroplastswere isolated. Whereas NADP-reduction under alkaline assay conditionsalways exhibits a stimulation upon addition of Mg⁺⁺, measurementsin an acidic medium show an age-dependent reversal to inhibition.The data for oxygen evolution are more complex in nature, dueto participation of consecutive reactions. The Mg⁺⁺ concentrationfor optimal ATP formation decreases with the age of the cotyledonarymaterial. The results are discussed from the viewpoint of apossible control of essential photosynthetic functions in vivoby magnesium.     

Studies on Isolated Protoplasts and Vacuoles: II. THE ACTION OF GROWTH SUBSTANCES

The response of isolated protoplasts to indol-3-yl acetic acidwas investigated, and they were found to undergo a rapid water-uptakewith ultimate rupture of the plasmalemma and release of thelarge central vacuole. The use of a photomicrographic methodshowed that this response was optimal at 10^-5 M indol-3-yl aceticacid. No such response could be detected for isolated vacuoles.¹⁴C-labelled indol-3-yl acetic acid was used to obtain furtherinsight into the site of action of this growth substance. Evidenceis presented which suggests that the site of action of indol-3-ylacetic acid, for this response, is the plasmalemma, where itfacilitates an increased uptake of solutes which is followedby an osmotic water uptake.     

Whole Plant Studies using Radioactive 13-Nitrogen: II. A COMPARTMENTAL MODEL FOR THE UPTAKE AND TRANSPORT OF NITRATE IONS BY Zea mays

Nitrate ion uptake by the roots of hydroponically grown maizeseedlings was measured using the short-lived isotope ¹³N. Itis shown to be described by a four compartment model, recognizablynitrogen in the root bathing solution, nitrogen which is readilyexchangeable from the root, nitrogen bound in the root, andnitrogen transported from the root. Some of the absorbed activity leaks back into the root bathingsolution with the efflux from the root, as a fraction of theinflux, increasing with concentration to be greater than 0–8at external nitrate ion concentrations above about 1.0 mol m^–3.The capacity of the exchangeable root pool increases with externalnitrate ion concentration, approaching the expected cytoplasmicnitrate ion content at the highest external nitrate ion concentrationsstudied (70 mol m^–3). The investigation has highlighted the problems of interpretinguptake profiles in experiments for which the 10 min half-lifeof ¹³N dictates experimental times that are comparable withthe times for saturation of root pools. Key words: Zea mays, ¹³N, Compartmental model, Nitrate uptake     

ACTIVE TRANSPORT BY THE CECROPIA MIDGUT : II. Fine Structure of the Midgut Epithelium

A morphological basis for transcellular potassium transport in the midgut of the mature fifth instar larvae of Hyalophora cecropia has been established through studies with the light and electron microscopes. The single-layered epithelium consists of two distinct cell types, the columnar cell and the goblet cell. No regenerative cells are present. Both columnar and goblet cells rest on a well developed basement lamina. The basal portion of the columnar cell is incompletely divided into compartments by deep infoldings of the plasma membrane, whereas the apical end consists of numerous cytoplasmic projections, each of which is covered with a fine fuzzy or filamentous material. The cytoplasm of this cell contains large amounts of rough endoplasmic reticulum, microtubules, and mitochondria. In the basal region of the cell the mitochondria are oriented parallel to the long axes of the folded plasma-lemma, but in the intermediate and apical portions they are randomly scattered within the cytoplasmic matrix. Compared to the columnar cell, the goblet cell has relatively little endoplasmic reticulum. On the other hand, the plications of the plasma membrane of the goblet cell greatly exceed those of the columnar cell. One can distinguish at least four characteristic types of folding: (a) basal podocytelike extensions, (b) lateral evaginations, (c) apical microvilli, and (d) specialized cytoplasmic projections which line the goblet chamber. Apically, the projections are large and branch to form villus-like units, whereas in the major portion of the cavity each projection appears to contain an elongate mitochondrion. Junctional complexes of similar kind and position appear between neighboring columnar cells and between adjacent columnar and goblet cells as follows: a zonula adherens is found near the luminal surface and is followed by one or more zonulae occludentes. The morphological data obtained in this study and the physiological information on ion transport through the midgut epithelium have encouraged us to suggest that the goblet cell may be the principal unit of active potassium transport from the hemolymph to the lumen of the midgut. We have postulated that ion accumulation by mitochondria in close association with plicated plasma membranes may play a role in the active movement of potassium across the midgut.     

Studies in Stomatal Behaviour: II THE ROLE OF STARCH IN THE LIGHT RESPONSE OF STOMATA PART 4. VARIATION UNDER CONSTANT CONDITIONS

A quantitative study has been made of the extent and natureof the variation in starch-content and aperture of the stomataof Pelargonium. It is shown that distribution over the surfaceof a single leaf is substantially normal in form; that underexperimental conditions in common use there is a highly significantdifference between groups of stomata on the lower epidermisof a single leaf; that the variation between different leaveson the same plant, or between similar leaves on different plants,does not differ significantly from that between strips fromthe same leaf; that not less than 20 stomata should be measuredon each strip; and that pore-width is a satisfactory measureof the area of the stomatal aperture.     

Studies on the Expansion of the Leaf Surface: II. THE INFLUENCE OF LIGHT INTENSITY AND DAYLENGTH

The parts played by constant amounts of visible radiation perday and its two components—daylength and intensity—ininfluencing the growth of Cucumis sativus have been investigated.The amount of radiation per day had a far greater influencethan either of its components per se. Nevertheless, small significanteffects of photoperiod were found, leaf expansion and dry weightincrease being greatest at daylengths between 10 and 15 hr.rather than with longer days which, with similar daily totals,would be expected to give the greatest amounts of assimilation. Rates of leaf production and appearance were greatest with thehighest amounts of radiation, but the rates of expansion ofindividual leaves and their maximum areas were greatest withintermediate amounts of radiation. This response resulted inan optimum curve relating the leaf surface and the dry weightattained after a given period to radiation. The amount of radiationgiving the maximum leaf surface and dry weight decreased withage and with external nutrient supply, but at any one age washigher for increase in dry weight than for increase in leafsurface; stem and root tissues responded more to high radiationthan did the leaf surface. The net assimilation rate was a linearfunction of visible radiation over the range of 15–120cal. cm.^-2 day^-1 explored, the highest value of radiation usedrepresenting the intensity at which photosynthesis would beexpected to be maximal over a 12–15 hr. day. The inhibitory effect of high radiation on leaf expansion andthe resultant influence on the growth of the plant are explainedin terms of the number and intensity of ‘sinks’for carbohydrate and mineral nutrients within the plant.     

INTRACELLULAR TRANSPORT OF SECRETORY PROTEINS IN THE PANCREATIC EXOCRINE CELL : II. Transport to Condensing Vacuoles and Zymogen Granules

In the previous paper we described an in vitro system of guinea pig pancreatic slices whose secretory proteins can be pulse-labeled with radioactive amino acids. From kinetic experiments performed on smooth and rough microsomes isolated by gradient centrifugation from such slices, we obtained direct evidence that secretory proteins are transported from the cisternae of the rough endoplasmic reticulum to condensing vacuoles of the Golgi complex via small vesicles located in the periphery of the complex. Since condensing vacuoles ultimately become zymogen granules, it was of interest to study this phase of the secretory cycle in pulse-labeled slices. To this intent, a zymogen granule fraction was isolated by differential centrifugation from slices at the end of a 3-min pulse with leucine-¹⁴C and after varying times of incubation in chase medium. At the end of the pulse, few radioactive proteins were found in this fraction; after +17 min in chaser, its proteins were half maximally labeled; they became maximally labeled between +37 and +57 min. Parallel electron microscopic radioautography of intact cells in slices pulse labeled with leucine-³H showed, however, that zymogen granules become labeled, at the earliest, +57 min post-pulse. We assumed that the discrepancy between the two sets of results was due to the presence of rapidly labeled condensing vacuoles in the zymogen granule fraction. To test this assumption, electron microscopic radioautography was performed on sections of zymogen granule pellets isolated from slices pulse labeled with leucine-³H and subsequently incubated in chaser. The results showed that the early labeling of the zymogen granule fractions was, indeed, due to the presence of highly labeled condensing vacuoles among the components of these fractions.     

Studies in Dormancy of Sycamore: II. THE EFFECT OF DAYLENGTH ON THE NATURAL GROWTH-INHIBITOR CONTENT OF THE SHOOT

1. An investigation was made into effect of daylength conditionsof the inhibition content of first-year seedlings of sycamore(Acer pseudoplatanus). 2. The shoot apical regions and mature leaves were extractedwith 80 per cent. Aqueous methanol, fractionated by paper chromatographyin isopropanol/ammonia and assayed by the wheat-coleptile growthtest. 3. A growth inhibitor was present in all extracts at R_f 0.7.Higher levels of inhibitor were present in both apices and matureleaves of plants transferred to short-day conditions than ofthose maintained under long-days throughout. 4. These difference in inhibitor level can be detected after2–5 days of short-day treatment, Preceding any markedeffect of daylength on growth. 5. Evidence is adduced in support of the hypothesis that theinhibitors is produced in the leaves during darkness and istransported to the apex during the photoperiod.     

Movement of C-Labeled Assimilates into Kernels of Zea mays L: II. Invertase Activity of the Pedicel and Placento-Chalazal Tissues

Invertases of the placento-chalazal and pedicel tissues are much more active than invertase from the pericarp of Zea mays L. kernels 12 to 40 days after pollination. Sucrose synthetase was not detected in the pedicel or placento-chalazal tissues. Sucrose content and percentage increased in the pedicel with advancing kernel age. Hexoses accounted for over half of the sugars extracted from the placento-chalazal tissues. These data are consistent with the hypothesis that sucrose translocated to the pedicel is hydrolyzed by acid invertase(s) prior to entry of sugar into the endosperm tissue. The placentochalazal tissue appears to be the primary site of sucrose inversion with the pedicel invertase contributing more or less to this process depending on kernel age.     

Studies on the Germination of the Seeds of Striga hermonthica: II. THE EFFECT OF THE STIMULATING SOLUTION ON SEED RESPIRATION

1. Data are presented which show the effect of the natural stimulatingsolution on the respiration of the seeds of Striga hermonthica.
2. The stimulating solution has been shown to enhance the aerobicrespiration of seeds exposed to it, compared with that of seedsto distilled water at the same temperature. This effect on therespiration of the seeds is quite independent of any previousmoisture-treatment of the seeds, and thus of germination.
3. Themaximum anaerobic rate of carbon-dioxide output of air-dryseedsafter treatment with the stimulating solution may be upto fifteentimes greater than that of seeds moistened with distilledwater.The anaerobic respiration of air-dry seeds after exposuretothe stimulating solution is greatly reduced by 0?025 M. sodiumfluoride and 0?001 M. sodium monoiodoacetate.
4. The stimulatingsolution has been found to have little effecton the anaerobicoutput of carbon dioxide from seeds which hadbeen moisture-treatedfor 6 days at 22? C. before treatmentwith the stimulating solution.
5. No correlation could be established between the effects ofthestimulating solution on germination and respiration.

     

Radial Water and Solute Flow in Roots of Zea mays: III. EFFECT OF TEMPERATURE ON THO AND ION TRANSPORT

THO influx, ³⁶Cl^– influx and efflux and ²²Na influx throughmaize cortex sleeves were assayed under different temperaturesranging from 5 to 40 °C. The highest Q¹⁰ and H values forTHO influx were found between 12 and 32 °C. ³⁶Cl effluxand ²²Na influx were almost temperature independent. ³⁶Cl influxwas greatly increased above 20°C. These results are consistentwith previous conclusions that water flow through maize sleevesis metabolically mediated and that chloride is actively pumpedinwards.     

Studies on the Infection,Colonization, and Movement of Pseudomonas syringae pv. actinidiae in Kiwifruit Tissues Using a GFPuv-Labeled Strain

Kiwifruit bacterial canker, an economically important disease caused by Pseudomonas syringae pv. actinidiae (Psa), has caused severe losses in all major areas of kiwifruit cultivation. Using a GFPuv-labeled strain of Psa, we monitored the invasion, colonization, and movement of the pathogen in kiwifruit twigs, leaves and veins. The pathogen can invade twigs through both wounds and natural openings; the highest number of Psa is obtained in cut tissues. We determined that, following spray inoculation, Psa-GFPuv could infect leaves and cause lesions in the presence and absence of wounds. Light and transmission electron microscopic observations showed that bacterial cells colonize both phloem and xylem vessels. Bacterial infection resulted in marked alterations of host tissues including the disintegration of organelles and degeneration of protoplasts and cell walls. Furthermore, low temperature was conducive to colonization and movement of Psa-GFPuv in kiwifruit tissues. Indeed, the pathogen migrated faster at 4°C than at 16°C or 25°C in twigs. However, the optimum temperature for colonization and movement of Psa in leaf veins was 16°C. Our results, revealing a better understanding of the Psa infection process, might contribute to develop more efficacious disease management strategies.