Spatial mapping of phosphorus influx in bean root systems using digital autoradiography

A novel technique was developed to spatially map the phosphorus net influx capacity in intact root systems. The method is based on digital autoradiography and permits the quantification of phosphorus influx at high spatial resolution (2 mm). Roots of 18-d-old common bean plants were exposed to (32)P-labelled orthophosphate, quickly frozen, excised, lyophilized, scanned, and exposed to a storage phosphor screen. Plots of (32)P content versus root length (distance from the root tip or from the base of the root) were obtained for three different root classes: basal, basal laterals, and taproot laterals. Radioactivity detected by filmless autoradiography correlated well (r(2)=0.99) with measurements made by scintillation counting. Basal roots absorbed 2.5 times and 1.9 times more phosphorus than the taproot lateral and basal lateral root classes, respectively, in the first 20 mm from the root apex. External phosphorus markedly affected influx: roots averaged 5, 16, and 34 pmol P min(-1) in the apical 20 mm when exposed to 1, 5, and 10 microM P solutions, respectively. The spatial pattern of phosphorus influx along the root axes of the different root classes was rather homogeneous when measured on a root surface area basis. Phosphorus influx in the older segments of basal roots (those next to the hypocotyl) did not differ from the newer segments close to the root apex. However, a heterogeneous pattern was detected for basal roots when measured on a length basis, indicating that both root class and diameter constitute main factors controlling the spatial pattern of net influx.     

A comparison of ammonium, nitrate and proton net fluxes along seedling roots of Douglas-fir and lodgepole pine grown and measured with different inorganic nitrogen sources

Significant spatial variability in NH4+, NO3- and H+ net fluxes was measured in roots of young seedlings of Douglas-fir (Pseudotsuga menziesii) and lodgepole pine (Pinus contorta) with ion-selective microelectrodes. Seedlings were grown with NH4+, NO3-, NH4NO3 or no nitrogen (N), and were measured in solutions containing one or both N ions, or no N in a full factorial design. Net NO3- and NH4+ uptake and H+ efflux were greater in Douglas-fir than lodgepole pine and in roots not exposed to N in pretreatment. In general, the rates of net NH4+ uptake were the same in the presence or absence of NO3-, and vice versa. The highest NO3- influx occurred 0-30 mm from the root apex in Douglas-fir and 0-10 mm from the apex in lodgepole pine. Net NH4+ flux was zero or negative (efflux) at Douglas-fir root tips, and the highest NH4+ influx occurred 5-20 mm from the root tip. Lodgepole pine had some NH4+ influx at the root tips, and the maximum net uptake 5 mm from the root tip. Net H+ efflux was greatest in the first 10 mm of roots of both species. This study demonstrates that nutrient uptake by conifer roots can vary significantly across different regions of the root, and indicates that ion flux profiles along the roots may be influenced by rates of root growth and maturation.     

Re-sorption of organic compounds by roots of Zea mays L. and its consequences in the rhizosphere

The exudation of soluble carbon compounds from Zea mays roots was investigated over a 10 day growth period under sterile and non-sterile solution culture conditions. The results showed that plants grown in sterile static solution culture, where C was allowed to accumulate, released 8 times less C than plants grown under culture conditions in which the solutions were replaced daily. The increased C loss from plant cultures in which exudates were removed daily was attributable to, (a) the reduced potential for root re-sorption of previously lost C, and (b), increasing diffusion gradients between the root and the surrounding bathing solution increasing passive leakage of exudates from the roots. In treatments where C was removed daily from the root-bathing solution, 86% of the total C lost was of a soluble low molecular weight nature, whereas, in sterile and non-sterile static cultures, allowing the accumulation of C over 10 days, this was reduced to 67.5 and 48% respectively. The main C fluxes operating in a solution culture system (efflux and influx of C by both roots and microorganisms) were examined using a computer simulation model to describe movement of soluble sugar-C in both sterile and non-sterile conditions. In sterile static cultures where C was allowed to accumulate in solution over a 10 day growth period, 98% of the C exuded was re-absorbed by the plant. Where C was removed daily from the root-bathing solution this was reduced to 86%. The predicted patterns of C accumulation were similar to those found in the experiments. Simulations showed that the pattern of accumulation and final equilibrium concentrations were dependent on the rate of exudation, the spatial characteristics of exudation, solution volume, root growth rate and the presence of a microbial population. Simulations under non-sterile conditions showed that roots can compete with microorganisms for exudates in solution indicating the possible importance of re-sorption in a soil environment. The results clearly indicate that roots are capable of regulating the net amount of C released into a solution culture with the amount of C collected being highly dependent on the experimental conditions employed. The possible implications of soluble C influx on processes operating within the rhizosphere and in experimental systems is discussed.     

Re-sorption of organic compounds by roots of Zea mays L. and its consequences in the rhizosphere

The influx and efflux of sugar-C and the cycling of C within intact maize roots (Zea mays L.) was studied in sterile solution culture. Using metabolic inhibitors it was shown that roots could take up sugars against the concentration gradient probably via H⁺-ATPase dependent plasmalemma proton cotransporters. In contrast to this, no evidence was found for an ATPase mediated efflux of sugars from the root. All parts of the root were capable of taking up exogenous sugars. Examination of sugar exudation sites along the root slowed efflux at all locations, with the amount of efflux linearly correlated with internal cellular concentration. The results clearly indicated that the influxefflux mechanisms are linked both spatially, temporally and with respect to the sugars capable of transportation. The turnover of C within the root was found to be extremely rapid with turnover of the soluble sugar pool being 0.8 to 15 times daily depending on root spatial location. The results strongly suggest that the recapture of sugars from outside the root plays an important role in regulating the amount of C lost to the soil which in turn will reduce both pathogen attraction and the size of the rhizosphere microbial population and will also increase the plant's C efficiency.     

Simulation of shoot chloride accumulation: separation of physical and biochemical processes governing plant salt tolerance

The effect of growth under saline condition (100 mol m^-3 NaCl in the nutrient solution) on the influx and the efflux of glucose from roots of cotton plants was analysed utilising the non metabolisable glucose analogue [¹⁴C]-3-O-methylglucose ([¹⁴C]3-OMG). Apical segments (1 cm long) excised from cotton roots took up [¹⁴C]3-OMG. At each tested concentration (5–500 mmol m^-3), the influx was completely inhibited by the presence of the protonophore carbonylcyanide-m-chlorophenyl hydrazone (CCCP) indicating that it is mediated by a H⁺-coupled co-transport mechanism. The CCCP-sensitive [¹⁴C]3-OMG influx was lower in the root segments excised from plants grown on saline solution than in the controls, and this was particularly evident at lower external concentrations. This difference was not due to a lower H⁺ apoplastic availability. In fact, the saline condition did not affect the pH of the rhizosphere and indeed the H⁺–ATPase activity, evaluated in plasma membrane vesicles purified from saline-treated plants, was higher (+23%) than in the controls. The lower uptake of [¹⁴C]3-OMG into saline treated root segments was related to an enhanced value of the apparent K_m of the carrier for the glucose analogue. This effect is discussed in relation to either the more positive value of the transmembrane electric potential difference (ΔΨ) measured in these root segments, or a competitive inhibition of Na⁺ on the H⁺ binding site of the carrier. Growth in saline solution slightly affected the efflux of the [¹⁴C]3-OMG preloaded in root segments, changing the membrane permeability to the molecule. The results strongly suggest that the higher (2.5 fold) net exudation of glucose, observed in short-term (4 h) collection experiments, from roots of cotton plants grown in saline condition, is mainly due to an effect of the saline growth condition on the system involved in the reabsorption of the hexose rather than on its efflux. This revised version was published online in June 2006 with corrections to the Cover Date.     

The contribution of different regions of the seminal roots of wheat to uptake of nitrate from soil

A technique was developed to determine the physiological activity of defined sections of seminal roots of wheat grown in sand. Wheat plants were grown for 2 weeks in narrow columns of N-deficient sand to which all other nutrients had been added. The columns were split longitudinally and ¹⁵N-labelled nitrate, in an agar medium, supplied to 2 cm sections of root. Shoots and roots were analysed after 24 h to determine the uptake of ¹⁵N. Three sections were examined on either the secondary or tertiary seminal root: 1 cm from the seed (basal segment), 35 cm from the seed (middle segment) and 4 cm from the root apex (apical segment). Total uptake was greatest from the basal and middle segments, declining by 50% from the apical segment. However, uptake per unit root length, including exposed sections of lateral roots, was not significantly different along the root.     

Aluminum Interactions with Voltage-Dependent Calcium Transport in Plasma Membrane Vesicles Isolated from Roots of Aluminum-Sensitive and -Resistant Wheat Cultivars

The role of Al interactions with root-cell plasma membrane (PM) Ca2+ channels in Al toxicity and resistance was studied. The experimental approach involved the imposition of a transmembrane electrical potential (via K+ diffusion) in right-side-out PM vesicles derived from roots of two wheat (Triticum aestivum L.) cultivars (Al-sensitive Scout 66 and Al-resistant Atlas 66). We previously used this technique to characterize a voltage-dependent Ca2+ channel in the wheat root PM (J.W. Huang, D.L. Grunes, L.V. Kochian [1994] Proc Natl Acad Sci USA 91: 3473-3477). We found that Al3+ effectively blocked this PM Ca2+ channel; however, Al3+ blocked this Ca2+ channel equally well in both the Al-sensitive and -resistant cultivars. It was found that the differential genotypic sensitivity of this Ca2+ transport system to Al in intact roots versus isolated PM vesicles was due to Al-induced malate exudation localized to the root apex in Al-resistant Atlas but not in Al-sensitive Scout. Because malate can effectively chelate Al3+ in the rhizosphere and exclude it from the root apex, the differential sensitivity of Ca2+ influx to Al in intact roots of Al-resistant versus Al-sensitive wheat cultivars is probably due to the maintenance of lower Al3+ activities in the root apical rhizosphere of the resistant cultivar.     

Citrate exudation from white lupin induced by phosphorus deficiency differs from that induced by aluminum

Both phosphorus (P) deficiency and aluminum (Al) toxicity induce root exudation of carboxylates, but the relationship between these two effects is not fully understood. Here, carboxylate exudation induced by Al in Lupinus albus (white lupin) was characterized and compared with that induced by P deficiency. Aluminum treatments were applied to whole root systems or selected root zones of plants with limited (1 microM) or sufficient (50 microM) P supply. Aluminum stimulated citrate efflux after 1-2 h; this response was not mimicked by a similar trivalent cation, La(3+). P deficiency triggered citrate release from mature cluster roots, whereas Al stimulated citrate exudation from the 5- to 10-mm subapical root zones of lateral roots and from mature and senescent cluster roots. Al-induced citrate exudation was inhibited by P limitation at the seedling stage, but was stimulated at later growth stages. Citrate exudation was sensitive to anion-channel blockers. Al treatments did not affect primary root elongation, but inhibited the elongation of lateral roots. The data demonstrate differential patterns of citrate exudation in L. albus, depending on root zone, developmental stage, P nutritional status and Al stress. These findings are discussed in terms of possible functions and underlying mechanisms.     

A Correlation between Structure and Function in the Root of Zea mays

The exudation rates of fluid and potassium ions from isolatedmaize roots were determined before and after excision of certainlengths of root tip. The results of this study suggest thatexcised maize roots possess the ability to absorb potassium(and presumably chloride) ions and concomitant amounts of waterover a considerable distance (10 cm) from the tip. Moreover,the observed power of absorption of ions and water into thetranslocatory pathway decreases in passing from the tip towardsthe base of the root. Both light and electron microscope techniques were used to examinethe anatomy of primary roots similar to those used in the physiologicalexperiments. The principal observation was that the xylem vesselsnear the root tip contain membrane-bounded cytoplasm with organelles.The number of mature xylem vessels, i.e. without cytoplasm,progressively increased in transverse sections cut from 1 to10 cm from the root tip; above 10 cm from the root tip all ofthe xylem vessels were found to be completely mature. It isevident that prima facie a connexion exists between this singleaspect of root anatomy and fluid exudation from excised roots. The uptake of tritiated water by roots and its transport intoexudates was examined. These data were analysed on the assumptionthat the exchange of external labelled water with the exudatewas achieved by the fluid exudation itself; this analysis indicatedthat an operational volume, similar to that of the total xylemvolume within the root, must become labelled during the formationof the exudate.     

How does nitrogen availability alter rhizodeposition in Lolium multiflorum Lam. during vegetative growth?

The objective of this work was to determine if the impact of nitrogen (N) on the release of organic carbon (C) into the soil by roots (rhizodeposition) correlated with the effect of this nutrient on some variables of plant growth. Lolium multiflorum Lam. was grown at two levels of N supply, either in sterile sand percolated with nutrient solution or in non-sterile soil. The axenic sand systems allowed continuous quantification of rhizodeposition and accurate analysis of root morphology whilst the soil microcosms allowed the study of ¹⁴C labelled C flows in physico-chemical and biological conditions relevant to natural soils. In the axenic sand cultures, enhanced N supply strongly increased the plant biomass, the plant N content and the shoot to root ratio. N supply altered the root morphology by increasing the root surface area and the density of apices, both being significantly positively correlated with the rate of organic C release by plant roots before sampling. This observation is consistent with the production of mucilage by root tips and with mechanisms of root exudation reported previously in the literature, i.e. the passive diffusion of roots solutes along the root with increased rate behind the root apex. We proposed a model of root net exudation, based on the number of root apices and on root soluble C that explained 60% of the variability in the rate of C release from roots at harvest. The effects of N on plant growth were less marked in soil, probably related to the relatively high supply of N from non-fertiliser soil-sources. N fertilization increased the shoot N concentration of the plants and the shoot to root ratio. Increased N supply decreased the partitioning of ¹⁴C to roots. In parallel, N fertilisation increased the root soluble ¹⁴C and the ¹⁴C recovered in the soil per unit of root biomass, suggesting a stimulation of root exudation by N supply. However, due to the high concentration of N in our unfertilised plants, this stimulation was assumed to be very weak because no significant effect of N was observed on the microbial C and on the bacterial abundance in the rhizosphere. Considering the difficulties in evaluating rhizodeposition in non sterile soil, it is suggested that the root soluble C, the root surface area and the root apex density are additional relevant variables that should be useful to measure along with the variables that are commonly determined when investigating how plant functioning impacts on the release of C by roots (i.e soil C, C of the microbial biomass, rhizosphere respiration).     

Iron absorption by wheat roots as a function of distance from the root tip

Summary The apex of the root has been proposed as the site of maximum nutrient uptake, a view based on the accumulation of some ions in the cells of apical meristem⁴. In this study, intact roots were grown across a thin agar sheet containing a radioisotope of iron. Uptake was then measured quantitatively by autoradiography of the dried agar once the root had been removed. Uptake patterns were not observed to occur around the roots of plants that had been supplied with sufficient iron. Around the roots of plants showing severe irondeficiency symptoms, patterns consistently showed depletion of iron in the agar starting at 3.5 cm behind the tip and continuing to the point of complete suberization of the endodermis. No uptake was observed around the apical 3.5 cm of the root, although the highest concentrations within the root have centered on the apex. Agronomy Paper No.869, New York State College of Agriculture.     

Effects of kinetin and root tip removal on exudation and potassium (rubidium) transport in roots of honey locust

Exudation, (86)Rb transport, and water permeability were examined in excised roots of honey locust (Gleditsia triacanthos L.) treated by removing the tip 2 mm (tip-cut 2 mm) or tip 8 mm of the root, or by adding kinetin, or by both treatments. Tip removal increased the rate of exudation. Kinetin, 5 x 10(-6)m, inhibited exudation and Rb transport in tip-cut 2-mm roots; the inhibition was reversible. Kinetin inhibition of exudation was initially associated with lower K(Rb) transport and later with decreases in both ion transport and water permeability. Exudation was also inhibited at 10(-10) to 10(-7)m kinetin. Exudation from roots with intact tips was not altered by kinetin until after about 24 hours. Light during the exudation period had no significant (95%) influence on rate of exudation during the first 24 hours whether root tips were cut or kinetin applied.The results suggest the involvement of the root tip in regulating exudation in other parts of the root. This regulation might occur through cytokinin control of water permeability and the rate of ion transport.     

Effect of pH and calcium on short-term NO3- fluxes in roots of barley seedlings

The effect of pH and Ca2+ on net NO3- uptake, influx, and efflux by intact roots of barley (Hordeum vulgare L.) seedlings was studied. Seedlings were induced with NO3- or NO2-. Net NO3- uptake and efflux, respectively, were determined by following its depletion from, and accumulation in, the external solution. Since roots of both uninduced and NO2(-)-induced seedlings contain little internal NO3- initial net uptake rates are equivalent to influx (M. Aslam, R.L. Travis, R.C. Huffaker [1994] Plant Physiol 106: 1293-1301). NO3-, uptake (influx) by these roots was little affected at acidic pH. In contrast, in NO3(-)-induced roots, which accumulate NO3-, net uptake rates decreased in response to acidic pH. Under these conditions, NO3- efflux was stimulated and was a function of root NO3- concentration. Conversely, at basic pH, NO3- uptake by NO3- and NO2(-)-induced and uninduced roots decreased, apparently because of the inhibition of influx. Calcium had little effect on NO3- uptake (influx) by NO2(-)-induced roots at either pH 3 or 6. However, in NO3(-)-induced roots, lack of Ca2+ at pH 3 significantly decreased net NO3- uptake and stimulated efflux. The results indicate that at acidic pH the decrease in net NO3- uptake is due to the stimulation of efflux, whereas at basic pH, it is due to the inhibition of influx.     

Graviresponsiveness of surgically altered primary roots of Zea mays

We examined the gravitropic responses of surgically altered primary roots of Zea mays to determine the route by which gravitropic inhibitors move from the root tip to the elongating zone. Horizontally oriented roots, from which a 1-mm-wide girdle of epidermis plus 2-10 layers of cortex were removed from the apex of the elongating zone, curve downward. However, curvature occurred only apical to the girdle. Filling the girdle with mucilage-like material transmits curvature beyond the girdle. Vertically oriented roots with a half-girdle' (i.e. the epidermis and 2-10 layers of the cortex removed from half of the circumference of the apex of the elongating zone) curve away from the girdle. Inserting the half-girdle at the base of the elongating zone induces curvature towards the girdle. Filling the half-circumference girdles with mucilage-like material reduced curvature significantly. Stripping the epidermis and outer 2-5 layers of cortex from the terminal 1.5 cm of one side of a primary root induces curvature towards the cut, irrespective of the root's orientation to gravity. This effect is not due to desiccation since treated roots submerged in water also curved towards their cut surface. Coating a root's cut surface with a mucilage-like substance minimizes curvature. These results suggest that the outer cell-layers of the root, especially the epidermis, play an important role in root gravicurvature, and the gravitropic signals emanating from the root tip can move apoplastically through mucilage.     

Aluminum activates a citrate-permeable anion channel in the aluminum-sensitive zone of the maize root apex. A comparison between an aluminum- sensitive and an aluminum-resistant cultivar

In search for the cellular and molecular basis for differences in aluminum (Al) resistance between maize (Zea mays) cultivars we applied the patch-clamp technique to protoplasts isolated from the apical root cortex of two maize cultivars differing in Al resistance. Measurements were performed on protoplasts from two apical root zones: The 1- to 2-mm zone (DTZ), described as most Al-sensitive, and the main elongation zone (3-5 mm), the site of Al-induced inhibition of cell elongation. Al stimulated citrate and malate efflux from intact root apices, revealing cultivar differences. In the elongation zone, anion channels were not observed in the absence and presence of Al. Preincubation of intact roots with 90 microM Al for 1 h induced a citrate- and malate-permeable, large conductance anion channel in 80% of the DTZ protoplasts from the resistant cultivar, but only 30% from the sensitive cultivar. When Al was applied to the protoplasts in the whole-cell configuration, anion currents were elicited within 10 min in the resistant cultivar only. La3+ was not able to replace or counteract with Al3+ in the activation of this channel. In the presence of the anion-channel blockers, niflumic acid and 4, 4'-dinitrostilbene-2, 2'disulfonic acid, anion currents as well as exudation rates were strongly inhibited. Application of cycloheximide did not affect the Al response, suggesting that the channel is activated through post-translational modifications. We propose that the Al-activated large anion channel described here contributes to enhanced genotypical Al resistance by facilitating the exudation of organic acid anions from the DTZ of the maize root apex.     

Stage-dependent border cell and carbon flow from roots to rhizosphere

Rising CO(2) levels in the atmosphere have drawn attention to the important role of soil in sequestering carbon. This project goal was to quantify soil carbon deposition associated with border cell release and exudation from root growth zones. Carbon was measured with a Carlo Erba C/N analyzer in soil from the rhizosphere of mature grasses and, in separate experiments, in soil collected around root growth zones. Root border cells in "rhizosphere soil" (silica sand) were counted using a compound microscope after soil sonication and extraction with surfactant. For sand-grown Bromus carinatus, Zea mays, and Cucumis sativus, young seedlings (with roots shorter than 2 cm) released thousands of border cells, while older root tips released only hundreds. For a variety of native annual and perennial grasses and invasive annual grasses (Nassella pulchra, B. carinatus, B. diandrus, B. hordeaceus, Vulpia microstachys, Aegilops triuncialis, Lolium multiflorum, Zea mays), the rhizosphere of mature root systems contained between 18 and 32 μg C g(-1) sand more than that of the unplanted controls. Spatial analysis of the rhizosphere around the cucumber growth zone confirmed C enrichment there. The root tip provided C to the rhizosphere: 4.6 μg C in front of the growing tip, with the largest deposition, 20.4 μg C, to the rhizosphere surrounding the apical 3 mm (root cap/meristem). These numbers from laboratory studies represent the maximum C that might be released during flooding in soils. Scaling up from the organ scale to the field requires a growth analysis to quantify root tip distributions in space and time.     

Rhizosphere carboxylate concentrations of chickpea are affected by soil bulk density

We investigated whether carboxylate exudation by chickpea (Cicer arietinum L.) was affected by soil bulk density and if this effect was local or systemic. We hypothesised that concentrations of carboxylates would increase with distance from the root apex due to continuous and constitutive accumulation of carboxylates, and that exudate accumulation would be greater in a compacted soil than in a loose soil. Plants were grown in split-root or single cylinders containing loose (1400 kg m (-3)) or compacted (1800 kg m (-3)) soil. Rhizosphere carboxylate concentrations were measured of whole root systems as well as of sections along the root. The root diameter was greatest of plants grown in the compacted soil; however, root diameters were the same for both root halves in the split-root design, whether they grew in loose soil or in compacted soil. Similarly, carboxylate concentrations tended to be lower for the whole root system in the compacted soil, but were the same for both root halves in the split-root design, irrespective of whether the roots were in loose soil or in compacted soil. These results indicate that both root diameter and carboxylate exudation by roots in chickpea is regulated systemically via a signal from the shoot rather than by local signals in the roots. There was no accumulation of carboxylates with increasing distance from the apex, probably because microbial degradation occurred at similar rates as carboxylate exudation. Malonate, previously suggested as deterrent to microorganisms, is likely only a selective deterrent.     

Root-hair response to vector-free gravity: role of Ca2+ ions

In plants, apical growth is demonstrated by a variety of cells, including root hairs (RH) which are tubular outgrowths of root epidermal cells. They are likely to be involved in uptake of nutrients and water, anchorage of plants, maintenance of contact between roots and soil, and root exudation. Over the last years, it has become clear that calcium is involved in various processes which result in tip growth. Previous studies from our laboratory have demonstrated an increase in calcium level in root cells of pea seedlings grown under conditions of space flight and clinorotation. On the basis of these data, we have suggested that such effects of microgravity and clinorotation might be due to the enhancement of Ca2+ influx into hyaloplasm evidently through Ca2+ channels. In this regard, it was interesting to examine the effects of clinorotation and Ca2+ channel blockers (verapamil and nifedipine) on orientation and structure of growing RH that might be an appropriate model system being sensitive to calcium for studying the gravitational effects at cellular level.     

Root length and biomass losses during sample preparation with different screen mesh sizes

Data are presented on the differences in root length density (RLD), dry matter (DM), and root diameter values determined on wheat and faba bean using sieves of different mesh size to separate roots from soil during sample preparation. Screens with 0.2, 1, and 2 mm (0.04, 1, and 4 mm²) aperture were used. Roots collected on the 2-mm sieve represented on average 55% of the weight and only 10% of the total length collected using a 0.2-mm sieve. With a 1-mm sieve 75% of weight was retained, but only 34% of the length. In the 0–20 cm soil layer average RLD and DM values ranged between 1.3 and 2.5 cm cm^-3 and 215 and 136 g m^-2 for faba bean and wheat respectively with 2 mm screens and 14.6 and 18.1 cm cm^-3 and 313 and 202 g m^-2 with 0.2 mm sieves. RLD was more affected than weight since losses from coarse screens were largely due to fine root fractions, although the 1-and 2-mm screens retained a small amount of fine roots that were long or attached to main structures. Variability was higher for measurements on coarser screens. The use of screens much coarser than the diameter of fine roots is not recommended for the study of surface-related phenomena in which root length quantification is necessary, while it may be acceptable for gross comparisons of root weight and spatial extent.     

Oscillatory chloride efflux at the pollen tube apex has a role in growth and cell volume regulation and is targeted by inositol 3,4,5,6-tetrakisphosphate

Oscillatory growth of pollen tubes has been correlated with oscillatory influxes of the cations Ca(2+), H(+), and K(+). Using an ion-specific vibrating probe, a new circuit was identified that involves oscillatory efflux of the anion Cl(-) at the apex and steady influx along the tube starting at 12 microm distal to the tip. This spatial coupling of influx and efflux sites predicts that a vectorial flux of Cl(-) ion traverses the apical region. The Cl(-) channel blockers 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino)benzoic acid completely inhibited tobacco pollen tube growth at 80 and 20 microM, respectively. Cl(-) channel blockers also induced increases in apical cell volume. The apical 50 micro m of untreated pollen tubes had a mean cell volume of 3905 +/- 75 microm(3). DIDS at 80 microM caused a rapid and lethal cell volume increase to 6206 +/- 171 microm(3), which is at the point of cell bursting at the apex. DIDS was further demonstrated to disrupt Cl(-) efflux from the apex, indicating that Cl(-) flux correlates with pollen tube growth and cell volume status. The signal encoded by inositol 3,4,5,6-tetrakisphosphate [Ins(3,4,5,6)P(4)] antagonized pollen tube growth, induced cell volume increases, and disrupted Cl(-) efflux. Ins(3,4,5,6)P(4) decreased the mean growth rate by 85%, increased the cell volume to 5997 +/- 148 microm(3), and disrupted normal Cl(-) efflux oscillations. These effects were specific for Ins(3,4,5,6)P(4) and were not mimicked by either Ins(1,3,4,5)P(4) or Ins(1,3,4,5,6)P(5). Growth correlation analysis demonstrated that cycles of Cl(-) efflux were coupled to and temporally in phase with cycles of growth. A role for Cl(-) flux in the dynamic cellular events during growth is assessed. Differential interference contrast microscopy and kymographic analysis of individual growth cycles revealed that vesicles can advance transiently to within 2 to 4 microm of the apex during the phase of maximally increasing Cl(-) efflux, which temporally overlaps the phase of cell elongation during the growth cycle. In summary, these investigations indicate that Cl(-) ion dynamics are an important component in the network of events that regulate pollen tube homeostasis and growth.