Effects on the Stem of Winter Barley of Manipulating the Source and Sink during Grain-filling: II. CHANGES IN THE COMPOSITION OF WATER-SOLUBLE CARBOHYDRATES OF INTERNODES

Field-grown barley plants were manipulated by removing earsand by shading to promote and to reduce, respectively, the storageof carbohydrates in their stems. Water-soluble carbohydrate(WSC) was extracted and separated by HPLC into glucose, fructose,sucrose, and fructan of degree of polymerization (DP) 3, 4,5, > 5, and measured in both the penultimate and fourth internodesfrom the ear to determine the effects of the manipulations. During the accumulation of WSC, the mass of fructan with a DPgreater than 5 continued to increase whilst the mass of fructansof DP 3 to 5 reached a maximum and then remained constant. Fructanaccumulated in internodes while they were extending althoughmost of the fructan in an internode accumulated after it wasfully extended. When WSC was mobilized from the stem, the massof glucose, sucrose and fructan decreased but the mass of fructosefirst increased then decreased, indicating that fructan washydrolysed at a faster rate than its product, fructose, couldbe utilized. Plants shaded to 50% of incident light from 14 d after anthesisaccumulated the same mass of WSC in the stem as controlplantsin one crop, whereas in another crop, plants shaded to 9% ofincident light from 11 d after anthesis accumulated less WSCthancontrol plants. WSC in the stem was lost from the more intenselyshaded plants earlier than from control plants. Plants de-eared at anthesis and at 21 d after anthesis accumulateda similar mass of WSC to control plants, although plants de-earedat 9 d after anthesis, in another crop, accumulated a greatermass of WSC than control plants. Although control plants mobilizedalmost all of their stored WSC, de-eared plants retained 43–55%of the WSC stored in their penultimate internode and did notlose any stored WSC from their fourth internode. In the penultimateinternodes of de-eared plants the mass of glucose, sucrose andfructan decreased and this was balanced in part by an increasein the mass of fructose. In the fourth internode, the mass ofWSC remained constant but the mass of accumulated fructose wasequal to the decrease in the mass of fructan. Results are discussed in relation to current knowledge of fructanmetabolism and to contributions of stored WSC to grainyield. Key words: Winter barley, water-soluble carbohydrate, fructan, de-earing, shading     

IPOMEAMARONE ACCUMULATION AND LIPID METABOLISM IN SWEET POTATO INFECTED BY THE BLACK ROT FUNGUS I. IDENTIFICATION OF STEROL AND CHANGES IN LIPID METABOLISM DURING INFECTION PROCESS

Changes in lipid contents during infection process in the non-infectedtissue adjacent to the infected region of diseased sweet potatoroots with black rot were examined in comparison with cut controltissue. Incorporation of 2 ¹⁴C-acetate and ³²P-phosphate intolipid fraction was also investigated. Although there was nosignificant change in lipid ester groups in both tissues, increasein phospholipids was found in diseased tissue. Sterol isolatedfrom fresh material was identified with ß-sitosterol.Chromatographic patterns of non-phospholipid fraction of diseasedtissue suggested that some metabolic alteration of this fractionmight occur in response to infection. ¹This paper constitutes Part 40 of Phytopathological Chemistryof Sweet Potato with Black Rot.     

CHANGES IN THE STABILITY AND POTENTIAL OF CELL SUSPENSIONS : I. THE STABILITY AND POTENTIAL OF BACTERIUM COLI.

1. Stability and potential of Bacterium coli suspensions depend, not only on the strain of the organism and the medium in which it is suspended, but also on the previous treatment of the suspension, and the length of time it has been in the medium. 2. When treated at acid reactions, the negative charge on the bacteria is diminished; with some strains, a positive charge is acquired. Changes in stability accompany the changes in potential. 3. Washing acid-treated bacteria at neutral or slightly alkaline reactions does not restore the original potential; the zone of flocculation is moved toward the alkaline side. 4. These changes are due to two factors: the extraction of a soluble protein which combines with the surfaces of the cells, and a further irreversible change of the cell or its membrane.     

Physiological Studies on Germination of Phaseolus mungo Seeds: I. DEVELOPMENT OF RESPIRATION AND CHANGES IN THE CONTENTS OF CONSTITUENTS IN THE EARLY STAGES OF GERMINATION

Changes in respiration rate and in the contents of various constituentsduring the early period of germination of Phaseolus mungo seedswere studied. The course of the respiration developed in threephases. A sharp rise was observed in the first phase (Phasea), followed by the second phase (Phase b) of fairly constantrespiration rate. The respiration rate increases again in thethird phase (Phase c). The O² uptake in Phases a and b was notinhibited by iodoacetate and fluoroacetate, while that in Phasec was inhibited. The contents of aspartic and glutamic acidsand amides were very high. A decrease of aspartic acid contentwas notable during the first few hours of germination. Citricand malic acids were the major organic acid constituents. Citricacid content in the seeds decreased, while that of malic acidremained unchanged. The leaching of malic acid into the soakingmedium was remarkable during the first 6 h of imbibition     

Effects of Shading the First Leaf on Growth of Barley Plants: I. Long-term Experiments

Plants of barley were grown under controlled conditions andthe first or second leaves covered with tubular shades thusreducing the light intensity at the leaf surface to low levels.Expansion of the shaded leaves was not prevented, but appearanceof the next leaf but one and all subsequent leaves on the mainstemwas delayed by up to 3 days. Primordia of the first four leaveswere present in the dry grain. Shade treatment delayed slightlythe initiation of the eighth and subsequent leaves and transitionto the double ridge stage at the mainstem apex. Shading the first leaf caused a temporary reduction in the rateof dry-matter increase of plants, but after 14 days the ratewas similar to that of control plants. Smaller effects werefound when the second leaf was shaded. Dry-matter productionfollowed two logarithmic phases in the period prior to awn emergence,and rates for the whole plant and for plant parts were similarfor control and shaded plants. Thus, apart from the initialperturbation, shading had no effect on growth in terms of rateof dry-weight gain. Shade treatment did not affect weight per grain or numbers ofgrain per ear, but over-all yield of grain was significantlyreduced since shading delayed the appearance of tillers andalso reduced the number of tillers bearing grain. The effectof shade was especially marked on tillers originating on primarytillers. Similar qualitative effects on tiller development werefound in an experiment on wheat.     

BIOGENESIS OF ETHYLENE IN APPLE TISSUE: I. FORMATION OF ETHYLENE FROM GLUCOSE, ACETATE, PYRUVATE, AND ACETALDEHYDE IN APPLE TISSUE

1. With the aim of elucidating the path of carbon in the formationof ethylene in plants, studies were made on the incorporationof ¹⁴C into ethylene evolved from apple slices, using several¹⁴C- labeled compounds as substrates. The effects of inhibitorswere also investigated. 2. The formation of ethylene-¹⁴C from glucose-¹⁴C was inhibitedby fluoride, but unaffected by arsenite, thus suggesting thatglucose is converted to ethylene via pyruvate. 3. Acetate is converted to ethylene after cleavage of C-l andC-2. Only a small portion of the latter (C-2) enters the moleculeof ethylene, the former (C-l) is detected in carbon dioxide.On the other hand, 2, and 3-carbons of pyruvate are converted,without splitting, to ethylene. 4. On removal of air, the incorporation of ¹⁴C into ethylenefrom acetate-2-¹⁴C was depressed, while that from pyruvate-¹⁴Cwas unaffected. 5. Acetaldehyde-l,2-¹⁴C is converted to ethylene without conversioninto ethanol. 6. These results are interpreted to suggest the occurrence ofthe pathway in which pyruvate and acetaldehyde may serve asprecursors of ethylene. ¹ A part of this paper was read at the regular Meeting of KansaiBranch of the Agricultural Chemical Society of Japan in Kyoto,October, 1964, and at the Annual Meeting of the Japanese Societyof Plant Physiologists in Tokyo, April, 1965 and presented ina preliminary form elsewhere (10).     

Defoliation-Induced Stress in Nodules of White Clover: I. CHANGES IN PHYSIOLOGICAL PARAMETERS AND PROTEIN SYNTHESIS

Nodule function and protein synthesis were studied in defoliationstressed white clover plants. Uncut control plants (C) werecompared with plants from two defoliation treatments: (1) continuousdefoliation (CD) where all leaves and petioles were removedeach day; and (2) defoliated/recovered (DR) where, after removalof all leaves and petioles, new leaves were then allowed toregrow. After a single defoliation N₂ fixation (acetylene reductionactivity) and nitrogenase-linked respiration declined by morethan 80% within 3 h and by nearly 100% by 24 h. DR plants beganto fix nitrogen again at a very low level 3 d later and thereafterrose to control levels by 15 d. Continuously defoliated plantsnever recovered N₂ fixation capacity. Nodule protein complementwas assessed by polyacrylamide gel electrophoresis. Major changesoccurred in buffer soluble protein band patterns by 6 d in CDplants, but few changes were evident in SDS soluble proteins.By 9 and 14 d significant disruption of all proteins was evident.The prominent host plant protein, leghaemoglobin (Lb) had disappearedby 14 d. In DR plants the intensity of staining was reducedbut no major changes in band patterns were evident and by 21d nodules were rejuvenated. [³⁵S]-labelled methionine was incorporated into nodule proteinsfrom all treatments throughout the experiment. However, continuousdefoliation caused increasing variability between replicatesin the labelled band patterns. By 21 d CD, much of the labelledprotein was present as amorphous low M_r material which suggestseither disruption of the protein synthesizing machinery or rapidhydrolysis by proteolytic enzymes. Surprisingly [³⁵S]-methionine was never found in Lb from nodulesof any treatment. It is possible that white clover Lb does notcontain any methionine residues or that no synthesis of Lb occurred. Key words: Trifolium repens, white clover, defoliation, protein synthesis, nodules     

Nitrogen Utilization in N-limited Barley during Vegetative and Generative Growth: I. GROWTH AND NITRATE UPTAKE KINETICS IN VEGETATIVE CULTURES GROWN AT DIFFERENT RELATIVE ADDITION RATES OF NITRATE-N

Growth and nitrate uptake kinetics in vegetatively growing barley(Hordeum vulgare L., cvs Laevigatum, Golf, and Mette) were investigatedin solution culture under long-term limitations of externalnitrogen availability. Nitrate was fed to the cultures at relativeaddition rates (RA) ranging from 0.02 to 0.2 d^–1. Therelative growth rate (RG, calculated for total plant dry weight)correlated well with RA in the range 0.02 to 0.07 d^–1.In the RA range from 0.07 to 0.2 d^–1 RG continued to increase,but an increasing fraction of nitrogen, added and absorbed,was apparently stored rather than used for structural growth.The RG of the roots was less affected by RA. V_max, for net nitrateuptake increased with RA up to 0.11 d^–1, but decreasedat higher RA. The decline in V_max coincided with a build-upof nitrate stores in both roots and shoots. V_max, expressedper unit nitrogen in the plants (the relative V_max, was higherthan required for maintenance of growth (up to 30-fold) at lowRA, whereas at higher RA the relative V_max decreased. Kineticpredictions of steady-state external nitrate concentrationsduring N-limited growth ranged from 0.2 to 5.0 mmol m^–3over the RG range 0.02 to 0.11 d_–1. It is suggested thatthe nitrate uptake system is not under specific regulation atlow RA, but co-ordinated with root protein synthesis and growthin general. At RA higher than 0.11 d_–1, however, specificregulation of nitrate uptake, possibly via root nitrate pools,become important. The three cultivars showed very similar growthand nitrate uptake characteristics. Key words: Barley, growth, nitrogen limitation, nitrate uptake, kinetics     

Nitrogen-13 Studies of Nitrate Fluxes in Barley Roots: I. COMPARTMENTAL ANALYSIS FROM MEASUREMENTS OF 13N EFFLUX

The short-lived radio-isotope nitrogen-13 (half-life 10 min)was used as a tracer in studying fluxes of N in the roots ofintact barley plants. After supplying the plants with ¹³N-nitratefor 30 min, efflux of ¹³N into an unlabelled (wash) solutionwas followed under steady-state conditions for a further 10min. Tests with ion exchange resins suggested that all of the¹³N released during this period was in the form of nitrate. In addition to nitrate from a surface film of solution and fromthe free space of the roots, efflux from another compartmentwas detected, tentatively identified as the cytoplasmic nitratepool. In plants grown with nitrate as the only external N-source,efflux from this compartment decreased with a rate constantabout 0·17 min^–1 (half-time 4 min). Adding ammoniumsulphate to the wash solution alone did not significantly affecteither the initial rate, or the rate constant, of efflux of¹³N from these roots. However, ¹³N efflux decreased more rapidly(rate constant about 0·32 min^–1, half-time 2·2min) in roots grown in, and subsequently washed with, solutioncontaining ammonium nitrate. In barley plants grown with 1·5 mol m^–3 nitrate,the cytoplasmic nitrate pool was estimated to contain about2% of the total nitrate in the roots, corresponding to a cytoplasmicnitrate concentration 26 mol m^–3. Nitrate efflux was equivalentto almost 40% of nitrate influx in the roots of these plants. Key words: Ion transport, nitrate, ammonium, efflux analysis, compartmentation     

Combined Effects of Partial Defoliation and Nutrient Availability on Cloned Betula pendula Saplings: I CHANGES IN GROWTH, PARTITIONING AND NITROGEN UPTAKE

The combined effects of partial defoliation and nutrient availabilityon dry matter accumulation and partitioning, and on nitrogenuptake and partitioning, were studied in cloned Betula pendulaRoth saplings. The saplings were randomly assigned to differentnutrient levels (5, 1·5 and 0·5 mol Nm^–3)in aerated nutrient culture and to the following defoliationtreatments: (1) control (no damage), (2)damage of the developingmain stem leaves (half of the leaf lamina removed), and (3)removalof the developing main stem leaves (entire leaf lamina removed).Measured in terms of cumulative whole-plant dry weight (includingremoved leaf tissue), the birch saplings were unable to compensatefor the loss of the developing leaves (treatment 3) during the14 d study period. In response to leaf removal (treatment 3)the mean final percentage reduction in whole-plant dry weightwas actually greater than the initial mean percentage reductioncaused by the removal itself; the magnitude of the final reductionwas independent of nutrient availability. After removal of thedeveloping leaves, branch growth was favoured at the expenseof the growth of the rest of the shoot; the relative branchgrowth was most pronounced at the highest nutrient level. Atthe two highest nutrient levels the nitrogen uptake of the saplingswith the developing leaves removed was less than that of undamagedsaplings. We suggest, however, that the incapacity of the saplingsfor compensatory growth after removal of the developing leaveswas primarily due to the decreased total carbon gain of thesaplings rather than to the decreased nitrogen uptake rate. Key words: Partial defoliation, nutrient availability, birch sapling, dry matter, nitrogen     

Effects of Mechanical Impedance on Root Growth in Barley (Hordeum vulgare L. ): I. EFFECTS ON THE ELONGATION AND BRANCHING OF SEMINAL ROOT AXES

Apparatus is described in which the root system of plants canbe exposed to accurately known external stresses in beds ofballotini for periods up to 3 weeks. A constant flow of aeratednutrient solution was supplied. Modifications of the apparatusallowed the emerging coleoptile also to be subjected to mechanicalstress. Earlier observations that roots are unable to decrease in diameterto enter small pores were confirmed. The effect of externalpressure on the rate of root elongation was independent of thediameter of pores provided this was smaller than the penetratingroot. Pressures of 20 and 50 kPa reduced the elongation of theseminal axes of barley by 50 and 80 per cent respectively. Significantreductions in root extension were also caused by 50 kPa or lessin more limited experiments with a number of other crop plants.The extension of the coleoptile and the first leaf respondedsimilarly to that of the roots. When the diameter of pores inthe rooting medium was intermediate between those of axes andlaterals, the former being impeded, an enhanced developmentof laterals led to a dense superficial root system. This considerablemodification of root form was without effect on nutrient uptakeprovided that an adequate supply of nutrients was availablein the restricted rooting zone. Attention is directed to some of the implications of these responsesfor plant growth under field conditions. However, considerationof the physiological mechanisms responsible for the effectsof low external pressures and the changes in cell structureto which they lead are deferred to subsequent papers.     

Photosynthetic Decline from High Temperature Stress during Maturation of Wheat : II. Interaction with Source and Sink Processes

High temperature stress reduces grain growth in wheat (Triticum aestivum L.) by altering source activity and sink capacity. The impact of stress on source and sink interactions in two wheat cultivars of differing source thermotolerance was monitored by analysis of chlorophyll fluorescence transients, Fv (variable fluorescence) and PSM (peak, stationary, maximum), of attached flag leaves on intact and decapitated tillers grown at optimum (20°C) and stress (35°C) temperatures after anthesis. The thermotolerant cultivar Waverly had reduced Fv and PS quenching and a large increase of SM during heat stress. The less thermotolerant cultivar, Len, exhibited increased Fv and PS quenching and a small increase of SM. Fluorescence induction was similar in intact and decapitated tillers of Len, indicating diminished sinksource interaction during heat stress. The present results and previous observations of photosynthetic activities indicate that cyclic electron transport and photophosphorylation in flag leaves of the thermotolerant cultivar were stimulated by sink demand (increased SM in intact plants). Reduced grain development in the thermolabile cultivar resulted from limited capacity to support cyclic electron transport and photophosphorylation (slight increase in SM of intact plants and large reduction of Cytochrome f/b₆-mediated electron transport capacity). It was concluded that heat stress injures the photosynthetic apparatus during reproductive growth of wheat and that diminished source activity and sink capacity may be equally important in reducing productivity.     

Nutrient Supply and the Growth of the Seminal Root System in Barley: I. THE EFFECT OF NITRATE CONCENTRATION ON THE GROWTH OF AXES AND LATERALS

A method was devised by which different zones along a singleseminal axis of an intact plant could be exposed for extendedperiods to contrasting concentrations of nitrate (either 0.01or 1.0 mM) in continuous flow, the supply of all other nutrientsbeing favourable throughout. The concentration of nitrate wasfound to exert a direct and strictly localized effect upon thegrowth of lateral roots which, depending upon the supply ofassimilates from the shoot, resulted in marked modificationsto the form of the root system. Zones receiving 1.0 mM nitrateshowed an increase in the number and extension rate of bothfirst- and second-order laterals, associated with a preferentialaccumulation of dry matter, compared with zones in 0.01 mM nitrate.The average number of laterals (both first and second order)per cm of parent root was 4.4 in the presence of 1.0 mM nitrateand 2.2 in 0.01 mM. The average extension rates of first-orderlaterals were 0.61 and 0.26 cm d^–1 and second-order laterals,0.10 and 0.05 cm d^–1 for nitrate concentrations of 1.0and 0.01 mM respectively. The precise numbers and extensionrates of laterals in any one zone were affected, however, bythe rate of growth of laterals in other parts of the root system.In contrast, the extension rates of axes were little affectedby the concentration of nitrate to which their apical meristemswere exposed and approached 2.0 cm d^–1 provided the plantswere not nitrogen-starved. The significance of these resultsto the physiology of root growth and soil-plant relations isdiscussed.     

Effects of Shading the First Leaf of Barley Plants on Growth and Carbon Nutrition of the Stem Apex

Effects of shading the first leaf on development of the apicalregion were investigated by examining the growth of leaf primordiaand the apical dome in the early seedling stages. Shade treatment affects the size of the dome; it was shown thatvalues for height, width, and volume of the dome of 12-day controlplants were always higher than for shaded plants. Primordialgrowth, in terms of length and dry weight, was reduced by shadeand growth in dry weight of the second, third, and fourth leaveswas shown to be dependent on photosynthetic production by thefirst leaf. Incorporation of ¹⁴C in the apical region was detected by autoradiographyon day 6 and increased with age. Transfer of assimilated carbonfrom the first leaf to the apex occurred during the first 3h after exposure to ¹⁴CO₂. On a unit dry-weight basis it isshown that the third and fourth leaves and apex incorporatedproportionately more labelled carbon than the larger older organssuch as the, second leaf. Shade treatment reduced incorporationinto the apical region and this is associated with the failureof the apex to grow over the period up to day 15. Evidence isprovided to show that in control plants the second leaf suppliescarbon to the apex from about day 12. The crucial importanceof the contribution of the first leaf to plant development isdiscussed.     

The Utilization of Cotyledonary Reserves in Phaseolus vulgaris L. cv. Carioca: I. CHANGES IN TOTAL AMYLOLYTIC AND PROTEOLYTIC ACTIVITY AND THE EFFECTS OF 6-BENZYLADENINE AND GIBBERELLIC ACID UPON WHOLE SEEDLINGS

Axis growth commenced only 20 h from imbibition in Phaseolusvulgaris when both uptake of water and oxygen were levellingoff. Cotyledonary dry material was exhausted by day 9, the greatestrates of transfer occurring from day 2. Utilization of reserveprotein proceeded relatively faster than dry matter and proportionatelymore was translocated to the shoot, particularly the leaf blades.Lamina protein and chlorophyll content showed a biphasic increasewith rapid synthesis prior to day 7 being followed by slowerrates up to maximum leaf area on day 17. The level of free aminoacids in the cotyledons fell continuously from day 3 and roseto peak levels in the axis between days 5 and 7. Soluble sugarsincreased throughout the period examined in the axis and accumulatedin the cotyledons prior to day 7. Exogenous application of GA₃ had marked morphological effectsupon the seedling though did not significantly alter the distributionof dry matter or protein reserves: 6-BA, in addition to distinctmorphological effects, delayed the mobilization of reservesto the axis. Total amylolytic activity in the cotyledon, optimal at pH 5.5,increased continually from day 3 to 9 whereupon activity abruptlydeclined. This increase was due to the appearance of -amylase;ß-amylase, while present, remained at a constant andcomparatively low level. Peak proteolytic activity occurredprior to that for amylases between days 5 and 7. 6-BA significantlyincreased both amylolytic and proteolytic activities in vitrothough did not alter the changes in levels of free amino acidsor sugars in the reserve tissue and axis. The discrepancy betweenin vivo and in vitro effects of 6-BA may be attributed to concomitanteffects upon seedling development whereby sink capacity is reduced.     

Effects of Iron and Oxygen on Chlorophyll Biosynthesis : I. IN VIVO OBSERVATIONS ON IRON AND OXYGEN-DEFICIENT PLANTS

Corn (Zea mays, L.), bean (Phaseolus vulgaris L.), barley (Hordeum vulgare L.), spinach (Spinacia oleracea L.), and sugarbeet (Beta vulgaris L.) grown under iron deficiency, and Potamogeton pectinatus L, and Potamogeton nodosus Poir. grown under oxygen deficiency, contained less chlorophyll than the controls, but accumulated Mg-protoporphyrin IX and/or Mg-protoporphyrin IX monomethyl ester. No significant accumulation of these intermediates was detected in the controls or in the tissue of plants stressed by S, Mg, N deficiency, or by prolonged dark treatment. Treatment of normal plant tissue with δ-aminolevulinic acid in the dark resulted in the accumulation of protochlorophyllide. If this treatment was carried out under conditions of iron or oxygen deficiency, less protochlorophyllide was formed, but a significant amount of Mg-protoporphyrin IX and Mg-protoporphyrin IX monomethyl ester accumulated.     

ISOLATION AND PROPERTIES OF SECRETORY GRANULES FROM RAT ISLETS OF LANGERHANS : I. Isolation of a Secretory Granule Fraction

A method has been devised for the isolation of a secretory granule fraction from isolated rat islets of Langerhans. The islets were homogenized in buffered sucrose, and the homogenate was separated into nuclear, mitochondrial, secretory granule, and microsomal fractions by differential centrifugation. The secretory granule fraction was purified by differential centrifugation in discontinuous sucrose density gradients. A greater degree of purification could be achieved by the use of two successive gradients of this type, although the final yield was greatly reduced. Biochemical and morphological characterization of the fractions was obtained; the secretory granule fraction contained both insulin and glucagon. The limiting membranes of the granules remained intact and the general appearance of the granules was similar to that seen within the whole islet cells.