Using Confocal Laser Scanning Microscopy to Measure Apoplastic pH Change in Roots of Lupinus angustifolius L. in Response to High pH

A confocal laser scanning microscopy method to measure apoplasticpH in situ of the tap roots of Lupinus angustifolius L. is described.Pseudo-ratiometric imaging of cell walls of lupin tap rootswas achieved by loading living roots with a pH-sensitive dye,Cl-NERF-dextran, and a pH-insensitive dye, Texas Red-dextran,as a reference. In 5-d-old tap roots which had previously beengrown at pH 5.4, the apoplastic pH in the elongating zone was5.2 ± 0.06. The apoplastic pH in roots grown at pH 7.5increased to 5.5. The method was validated by supplying differentforms of nitrogen in the nutrient solution. With     

Redistribution of Nutrient Elements from Cotyledons of Two Species of Annual Legumes During Germination and Seedling Growth

The distribution of dry matter and various mineral nutrientsbetween testa and embryo of seeds of Lupinus albus and L. angustifoliusis described It was found that lupin seeds at either end ofa pod contained less dry matter and minerals than seeds in themiddle of the fruit. The transport of dry matter, N, P, K, S,Ca, Mg, Na, Fe, Zn, Mn and Cu from cotyledons of parent seedsof both species to the seedling axis was measured from germinationto the time of cotyledon death. N, P, K and S were retrievedfrom cotyledons with over 90 per cent efficiency, dry matter,Mg, Na, Fe, Zn, Mn and with 59–90 per cent efficiency,and Ca with 26–31 per cent efficiency. There was littlechange in the efficiency of nutrient retrieval from cotyledonswhen seedlings were grown in different culture media. Both speciesshowed a linear relationship between the loss of each elementand dry matter throughout the experiment, and a similar proportioningbetween root and shoot of the amount of a specific nutrientmobilized from cotyledons of parent seeds. Lupinus albus L., Lupinus angustifolius L., lupin, transport, of dry matter and mineral nutrients     

Physiological responses of lupin roots to high pH

High pH seems to be a major constraint limiting the production of narrow-leafed lupin (Lupinus angustifolius L.) on alkaline soils. Whereas there has been much interest in soil acidity, relatively little is known about the effect of high pH on the growth of roots of higher plants.Elongation of roots of L. angustifolius was particularly sensitive to pH6.0 compared with other species. The effect of high pH in decreasing root elongation in L. angustifolius occurred within one hour. It was via an effect on cell elongation and not cell division and the effect was readily reversible. The mechanisms of the adverse effect of high pH are unknown. The permeability ratio of K⁺ to Na⁺ in the plasma-membrane of the root cortical cells was similar in solutions of both low pH and high pH. Reduced cell growth at high pH was not associated with an inefficiency of proton extrusion to the bulk solution by roots of this species. Nevertheless, increasing buffer concentration in the external solution decreased root elongation more in L. angustifolius than in Lupinus pilosus and Pisum sativum.     

Soil Water Extraction, Measured by Computer-Assisted Tomography, in Seedling Lupinus angustifolius cv. Yandee when Healthy and Infected with Phytophthora cinnamomi

The water-mould fungus Phytophthora cinnamomi Rands causes drought-likesymptoms on many hosts, and yet the mechanisms by which infectionleads to wilting are not fully understood. This is the firststudy to describe in detail changes in soil water around theroot with infection. Computer-assisted tomography (CAT) wasused with Lupinus angustifolius L. cv. Yandee to examine drawdowns(removal of soil water) around a central root infected by P.cinnamomi in a white sand. No growth differences in roots or shoots were found betweenhealthy and diseased plants during the 8 d of the experiment.However,drawdowns failed at high levels of inoculum (8–16 /Pc-infectedmillet seeds/plant) by 8 d. Water contents in pots with uninfectedplants were in the range 0·09–0·12 cm³ watercm^–3 soil in the centre of the pot, while water contentsin pots with infected plants at 16 millet seeds applied werein the range of 0·16–0·19 cm³ water cm^–3soil in the centre of the pot. A higher transpirational demand produced lower soil water contentsnear the root but this effect was confounded with infection:disease was more pronounced with higher transpirational demand,and disease led to an increase in water content. Key words: Root disease, Phytophthora cinnamomi, water uptake, soil-root interface, computer-assisted tomography     

Increased Synthesis of ABA in Partially Dehydrated Root Tips and ABA Transport from Roots to Leaves

Zhang, J. and Davies, W. J. 1987. Increased synthesis of ABAin partially dehydrated root tips and ABA transport from rootsto leaves.—J. exp. Bot. 38: 2015–2023. Isolated root tips of pea (Pisum sativum L. cv. Feltham First)and Commelina communis L. were air-dried until they lost between10% and 40% of their fresh weight, followed by a period of incubationat these reduced water contents. These treatments resulted inincreased ABA production, suggesting that root tips of bothspecies have the capacity to synthesize ABA in increased amountswhen water deficits develop in the root. The ABA concentrationin pea roots increased linearly as turgors fell below about0·15 M Pa and relative water contents (R WC) fell below90%. Commelina roots produced more ABA when RWC fell below asimilar value but the threshold turgor for increased ABA productionin Commelina roots was around 0·30 MPa. Roots of intact plants loaded with ABA as a result of incubationin solutions of varying concentrations provided ABA to leaveswhich resulted in increased ABA concentrations in the leaveswhen these were assayed several hours later. This occurred whenthese roots were not contributing substantially to transpirationalflux. Leaves on shoots that were enclosed and darkened and thereforenot transpiring, did not accumulate ABA from ‘loaded’roots. A role for root-sourced ABA in root-to-shoot communication ofthe effects of soil drying is discussed. Key words: ABA, roots, water relations     

Changes in the Root System of Wheat Seedlings Following Root Anaerobiosis I. Anatomy and Respiration in Triticum aestivum L.

Anatomical changes in roots of wheat seedlings (Triticum aestivumL. cv. Hatri) following oxygen deficiency in the rooting mediumwere investigated. The response of the plant to stress was testedat a very early developmental stage when the first adventitiousroots had just emerged. In order to analyze the adaptation ofdifferent roots, respiration rates of the roots 1–3 and4–n were compared with the respiration rates of the totalroot system. Oxygen deficiency was induced either by flushingnutrient solution with nitrogen or flooding of sand. In contrast to plants grown in well aerated media, both stressvariants led to a significant increase of the intercellularspace of the root cortex in seminal and first adventitious roots.Radial cell enlargement of cortical cells near the root tip,cell wall thickenings in flooded sand cultures and an increasein phloroglucinol-stainable substances were found to be furtherindicators of low oxygen supply. The roots 4–n which were promoted in growth under hypoxiashowed higher respiration rates; hence the total root respirationwas not restricted. Triticum aestivum L. cv. Hatri, wheat, roots, anatomy, anaerobiosis, stress, root respiration, intercellular space     

Characterization of a Non-abscission Mutant in Lupinus angustifolius L.: Physiological Aspects

A single-gene recessive mutant (Abs^-) of Lupinus angustifoliusL. ‘Danja’ that does not abscise any organs wascompared with its parent during continuous exposure of explantsfrom 14 d old seedlings to 10 µl l^-1ethylene. Both endo-(1,4)-ß- D -glucanase (cellulase) and polygalacturonase(PGA) activities increased significantly and progressively inpetiole-stem abscission zones of the parent before the onsetof abscission, and were reflected in a rapid decline in breakstrengthfrom 300 to 70 g within 32 h. In the mutant there was negligibleincrease in hydrolytic enzyme activity, breakstrength declinedslowly (to 180–200 g by 72 h) and there was no abscission.Isoelectric focusing showed two cellulase isoforms (pI 5.0 andpI 8.5) expressed in abscission zones of the parent; these wereexpressed at much lower levels in the mutant. These data areinterpreted to indicate that expression of at least two formsof cellulase activity is enhanced by ethylene in normal petioleabscission zones of lupin. PGA activity also increased in theabscission zone tissue of the parent but to a lesser extentin that of the mutant. We attribute the Abs^-phenotype to mutationof a gene regulating ethylene-responsive expression of abscission-specifichydrolytic enzymes. Copyright 2001 Annals of Botany Company Lupinus angustifolius, abscission, breakstrength, cellulase, ethylene, legume, lupin, mutant, polygalacturonase     

pH above 6.0 reduces nodulation in Lupinus species

Lupinus angustifolius and Lupinus pilosus differ substantially in root growth in response to high solution pH with L. angustifolius showing much greater sensitivity to pH above 6. This study examined the effect of pH above 6 on nodulation of these two species in buffered solution. Shoot weight and root weight and length in L. pilosus was not significantly affected by pH, whereas the growth of shoots and roots of L. angustifolius was markedly impaired by increasing pH. Total root length, the number of lateral roots, and the length of individual lateral roots were greatly decreased, resulting in decreased uptake of iron and phosphorus. In addition, L. angustifolius had a higher internal requirement for iron than L. pilosus. A solution pH above 6 decreased the number of nodule initials and nodules similarly in both species but decreased nodule mass much more in L. angustifolius. The effect of high pH on nodule formation occurred prior to that on host shoot growth. High pH also decreased nitrogen concentration and content in both species but to a greater extent for L. angustifolius. The results suggested that pH above 6 has a specific effect in the impairment of nodulation in lupins.     

Lupinus species differ in their requirements for iron

The effect of iron supply on the growth and nodulation ofLupinus angustifolius L. (Gungurru),Lupinus luteus L. (R-1171) andLupinus pilosus Murr. (P20957) was studied in acid solutions. Plants of the three species were grown together in the same solution and inoculated withBradyrhizobium (Lupinus) WU 425. Plants were then grown with or without applied NH₄NO₃. The lupin species differed greatly in their sensitivity to low iron concentrations in solution withL. pilosus being most tolerant andL. luteus most sensitive.L. pilosus had the highest iron concentration in tissues and had a higher ratio of iron concentration in the youngest fully expanded leaf blades (YEB) to that in roots than the other two species.L. luteus had higher iron concentrations in roots but lower iron concentration in YEB and shoots than didL. angustifolius. The requirements of internal iron for the maximal chlorophyll synthesis in YEB were 65 μg g^-1 forL. angustifolius andL. luteus, and 52 μg g^-1 forL. pilosus. In contrast to effects on growth, the three species had similar external iron requirements for nodule formation in roots and for maximal nitrogen concentrations in shoots. The results indicate that iron tolerant lupin species require lower internal and external iron supply and have a greater ability than sensitive species to translocate iron from roots to shoots.     

Growth and Development of Roots of Grapevine (Vitis vinifera L.) in Relation to Water Uptake from Soil

Developmental patterns of lateral roots and their vascular differentiationwere investigated for Vitis vinifera L. cv. Shiraz to assessthe likely contribution of lateral roots to total water uptakeof plants subjected to different irrigation regimes. Correlationanalyses showed a significant positive correlation between mainroot diameter and the diameter of first order lateral rootsof well-watered plants, but in water-stressed plants the twowere not significantly correlated. The correlations betweendiameters of first order lateral roots and the diameters ofmain roots were greater than correlations between the lengthsof first order laterals and the diameters of main roots. Thesuberised surface area of well-watered main roots increasedfrom 4% of total surface area at 0·25 cm to 100% at 10cm from the tip, whereas that of stressed plants increased from15% at 0·25 cm to 100% at 5 cm from the tip. In all treatmentsthe highest linear density of first order laterals was about7 laterals cm^-1 of main root. More than 50% of first order lateralshad diameters less than 0·05 cm, and more than 90% ofthem had lengths less than 5 cm. Calculations of axial resistancesbased on xylem diameter measurements suggest that the axialresistances of root segments may not be uniform along rootsas is often assumed in models of water uptake. Water flow intothe main roots via the lateral root pathway is likely to bemuch smaller than that via the direct radial flow pathway asonly about 1% of surface area of main roots is directly occupiedby lateral roots, leaving the other 99% of main root surfacearea available for the direct radial flow pathway.Copyright1994, 1999 Academic Press Axial resistance, grapevine (Vitis vinifera L. cv. Shiraz) roots, root diameter, root length, xylem vessels     

Unidirectional Ca2+ Fluxes in Roots of Rye (Secale cereale L). A Comparison of Excised Roots with Roots of Intact Plants

The unidirectional Ca²⁺ fluxes across the plasma membrane andtonoplast were determined in both excised roots and roots ofintact seedlings of rye (Secale cereale L. cv. Rheidol). Theunidirectional Ca²⁺ fluxes across the plasma membrane and tonoplastmeasured in excised roots were of a similar order of magnitudeto those determined in roots of intact plants. Influx and effluxof Ca²⁺ across the root plasma membrane were similar (estimatedto be between 0·7 and 3·4 µmol g     

The Role of the Stem in Phloem Loading of Minerals in Lupinus albus L. cv. Ultra

Collections of phloem sap made over a 40-day period from a varietyof locations on nodulated white lupin plants (Lupinus albusL. cv. ultra) showed considerable enrichment with K⁺ and Mg²⁺in the phloem streams destined for the shoot apices or fruitsrelative to the streams arising from the leaflets (up to 5.5times). Sodium showed enrichment in the streams destined forthe roots (up to 2.5 times) but only when present in the watersupply at a high level (3 mM). The stem, in view of its centrallocation in the transport pathway, is seen as an organ capableof redistributing minerals in the phloem independently of photosynthate. Lupinus albus L., lupin, phloem loading, magnesium, potassium, sodium, mineral elements     

Internal Root Anatomy of Maize Seedlings (Zea mays L.) as Influenced by Temperature and Genotype

The objectives of this investigation were to determine: (a)the general effect of temperature on internal root anatomy;(b) whether genotypic differences in such root traits exist;and (c) the association between internal root traits and shootgrowth, lateral root branching and cold tolerance of maize (Zeamays L.). Seedlings of 20 central European hybrids were grownunder high or low temperature (25/22·5 °C or 15/12·5°C day/night temperatures) until the third leaf was fullyexpanded. Light microscopy of cross sections revealed a largerdiameter of primary roots at low temperature which was due toa larger stele diameter and a thickening of the cortex. Concurrently,an increase in total cross sectional area of metaxylem elementswas obtained. It is assumed that the modification of the internalroot structure by temperature has an effect on both axial andradial water flow capacity. For all anatomical traits studied,variability between genotypes was apparent under both growingconditions. Furthermore, different genotypic responses to temperaturewere observed. However, basic differences between cold-tolerantand cold-sensitive genotypes did not exist. While at high temperatureroot traits and shoot growth were significantly and positivelycorrelated, at low temperature the correlation coefficient wasinsignificant. Consequently, it was not possible to characterizethe performance of the shoot at low temperature based on anatomicaltraits of the root. Moderate, positive correlation coefficientswere obtained between internal root traits and lateral rootbranching. The potential use of root anatomical traits as indirectselection criteria is discussed. Chilling tolerance, genotypes, root anatomy, Zea mays L     

Nitrogen Deficiency Slows Leaf Development and Delays Flowering in Narrow-leafed Lupin

Effects of nitrogen (N) supply on leaf and flower developmentinLupinus angustifolius L. cv Merrit were examined in a temperature-controlledglasshouse. Low N supply (0.05 or 0.4 mM N) had little effecton leaf initiation but slowed leaf emergence on the main stemcompared with plants receiving high N supply (6.0 or 6.4 mMN), or with symbiotic N₂-fixation. Plants experiencing transientN deficiency had slower leaf emergence than plants with a continuoussupply of 6.4 mM N. Nitrogen supply did not affect the timeof floral initiation, which occurred within 4 weeks of sowing,by which time nine to ten leaves had emerged. However, the floweringof low-N plants was delayed by 68 to 220 °C d (i.e. 4–14d) even though they had fewer leaves. The effect of N deficiencyon flowering time was largely a result of slower leaf emergence. Lupinus angustifolius L.; nitrogen; leaf; flower initiation; thermal time; plastochron; phyllochron     

Oxygen Diffusion in Lupin Nodules: I.VISUALIZATION OF DIFFUSION BARRIER OPERATION

Root nodules of Lupinus albus (L.) cv. Multolupa were subjectedto short- and medium-term stresses by lowering rhizosphere temperaturefrom 25 to 16°C (2 h), detopping plants (3 h), darkeningplants (21 h) or exposing roots to 20 mol m^–3 KNO₃ for4 d. All experimental treatments produced increases in oxygendiffusion resistance, compared with control plants. These correlatedwith structural changes in the nodule cortex, which is describedin detail for the first time. The most noticeable change isthe occlusion of intercellular spaces by a glycoprotein whichwas identified using the monoclonal antibody MAC236. This glycoproteinwas also found surrounding bacteria in intercellular spacesof the cortex of control nodules. Key words: Oxygen diffusion resistance, glycoprotein, nodules, nitrogen fixation, Lupinus albus     

Physiological Aspects of Cluster Root Function and Development in Phosphorus-deficient White Lupin (Lupinus albus L.)

Cluster root formation in white lupin (Lupinus albus L.) isinduced mainly by phosphorus (P) starvation, and seems to beregulated by the endogenous P status of the plant. Increasedformation of cluster roots, when indole acetic acid is suppliedto the growth medium of P sufficient plants, and inhibitoryeffects of kinetin application suggest the involvement of endogenousphytohormones (auxins and cytokinins), which may act in an antagonisticmanner in the P-starvation response. Phosphorus deficiency-inducedadaptations of white lupin, involved in P acquisition and mobilizationof sparingly available P sources, are predominantly confinedto the cluster roots, and moreover to distinct stages duringtheir development. Increased accumulation and exudation of citrateand a concomitant release of protons were found to be mainlyrestricted to mature root clusters after prolonged culture (3–4weeks) under P-deficient conditions. Inhibition of citrate exudationby exogenous application of anion channel antagonists such asethacrynic- and anthracene-9-carboxylic acids may indicate involvementof an anion channel. Phosphorus deficiency-induced accumulationand subsequent exudation of citric acid seems to be a consequenceof both enhanced biosynthesis and reduced turnover of citricacid in the cluster root tissue, indicated by enhanced expressionof sucrose synthase, fructokinase, phosphoglucomutase, phosphoenol-pyruvatecarboxylase, but reduced activity of aconitase and slower rootrespiration. The release of acid phosphatase and of phenoliccompounds (isoflavonoids) as well as the induction of a putativehigh-affinity P uptake system was more highly expressed in juvenile,mature and even senescent cluster regions than in apical zonesof non-proteoid roots. An AFLP-cDNA library for cluster root-specificgene expression was constructed to assist in the identificationof further genes involved in cluster root development. Copyright2000 Annals of Botany Company Acid phosphatase, auxin, citric acid, cluster roots, cytokinin, Lupinus albus L., P acquisition, P uptake, root exudates     

Roles of Morphology, Anatomy, and Aquaporins in Determining Contrasting Hydraulic Behavior of Roots

The contrasting hydraulic properties of wheat (Triticum aestivum), narrow-leafed lupin (Lupinus angustifolius), and yellow lupin (Lupinus luteus) roots were identified by integrating measurements of water flow across different structural levels of organization with anatomy and modeling. Anatomy played a major role in root hydraulics, influencing axial conductance (L_ax) and the distribution of water uptake along the root, with a more localized role for aquaporins (AQPs). Lupin roots had greater L_ax than wheat roots, due to greater xylem development. L_ax and root hydraulic conductance (L_r) were related to each other, such that both variables increased with distance from the root tip in lupin roots. L_ax and L_r were constant with distance from the tip in wheat roots. Despite these contrasting behaviors, the hydraulic conductivity of root cells (Lp_c) was similar for all species and increased from the root surface toward the endodermis. Lp_c was largely controlled by AQPs, as demonstrated by dramatic reductions in Lp_c by the AQP blocker mercury. Modeling the root as a series of concentric, cylindrical membranes, and the inhibition of AQP activity at the root level, indicated that water flow in lupin roots occurred primarily through the apoplast, without crossing membranes and without the involvement of AQPs. In contrast, water flow across wheat roots crossed mercury-sensitive AQPs in the endodermis, which significantly influenced L_r. This study demonstrates the importance of examining root morphology and anatomy in assessing the role of AQPs in root hydraulics.     

The Development of Desiccation-tolerance and Maximum Seed Quality During Seed Maturation in Six Grain Legumes

‘Physiological maturity’, i.e. the time when seedsreach their maximum dry weight during development, occurredwhen maturation drying on the parent plant in the field hadreduced seed moisture content to approximately 60 per cent infaba bean (Vicia faba L.), lentil (Lens culinaris Medic.), chickpea(Cicer arietinum L.), white lupin (Lupinus albus L.), soya bean(Glycine max [L.] Merr.) and pea (Pisum sativum L.) The onsetof desiccation-tolerance, i.e. the ability of seeds to germinatefollowing harvest and rapid artificial drying, coincided withphysiological maturity, except in pea where it occurred a littleearlier at about 70 per cent moisture content. Maximum seedquality as determined by maximum viability, minimum seedlingabnormalities and maximum seedling size occurred in pea, chickpeaand lupin when seeds were harvested for rapid drying at physiologicalmaturity; but for maximum seed quality in the other speciesmaturation drying had to proceed further - to about 45 per centmoisture content in soya bean and to about 30 per cent moisturecontent in lentil and faba bean seed crops. Much of this variationamongst the six species, however, was due to differences inthe variation in maturity within each seed crop. Results forindividual pods showed that peak maturity, i.e. maximum seedquality following harvest and rapid artificial drying, was achievedin all six species once maturation drying had reduced the moisturecontent of the seeds to 45–50 per cent. In pea, faba beanand soya bean there was a substantial decline in viability andan increase in seedling abnormalities when harvest was delayedbeyond the optimal moisture content for harvest.     

Pea Root Regeneration After Tip Excisions at Different Levels: Polarity of New Growth

The roots of light-grown pea seedlings (Pisum sativum L. cv.Alaska) were excised at 250, 500 or 1500 µm from the body/capjuncture. Tips were sampled for 7 d after excision to monitorthe polarity and structure of the regeneration response. Inroots excised at 250 µm a new single apex regeneratedwithout swelling or any sign of repair. The new root cap appearedidentical to the control. After the 500 µm excision tworesponses occurred. In 45% of the roots examined, a single newapex regenerated, in 55% two or three new apices regeneratedto form a dichotomized or trichotomized root in the same longitudinalaxis as the primary root. In roots excised at 1500 µm,one, two or three lateral roots formed adjacent to the xylemat right angles to the primary root axis. In most instancesthe new roots developed triarch xylem. The discussion concentrateson the relationship of the differentiation state of the vascularcylinder and the pericycle, at the excision site, to the regenerationresponse. Root regeneration, Pisum sativum, Pericycle, Root apex     

Ion Transport to the Xylem in Aerenchymatous Roots of Zea mays L.

Aerenchyma was induced in the developing nodal (adventitious)roots of maize (Zea mays L. cv. LG11) in solution culture bytreatment with an atmosphere of 5% (v/v) oxygen (oxygen partialpressure 5.0 k Pa). Ion uptake by aerenchymatous and ordinary(non-aerenchymatous) roots was then compared to determine theeffect of degeneration of much of the cortex in aerenchymatousroots on radial ion movement to the xylem. Rates of uptake andtransport of phosphate, potassium and chloride were followedby labelling 4–12mm length segments of intact roots usingradioactive tracers. In some experiments, enhanced rates ofphosphate transport were induced by means of a divided rootsystem and nutrient deprivation. For all three nutrient ions,and over a range of concentration for phosphate (10–100mmol m^–3 and potassium (0·25–5·0 molm^–3 and with enhanced rates of transport, aerenchymatousroots were at least as effective as ordinary roots (per mm³of root) and sometimes more effective (per mm root length).These findings are discussed in relation to hypotheses concerningthe pathways for radial ion movement across the cortex in ordinaryand aerenchymatous roots. Correspondence to: Long Ashton Research Station, Long Ashton,Bristol BS18 9AF, U.K. Key words: Aerenchyma, ion transport, mineral nutrition, oxygen deficiency, radio-isotopic tracers, roots, structure, Zea mays