Cell-specific localization of Na+ in roots of durum wheat and possible control points for salt exclusion

Sodium exclusion from leaves is an important mechanism for salt tolerance in durum wheat. To characterize possible control points for Na(+) exclusion, quantitative cryo-analytical scanning electron microscopy was used to determine cell-specific ion profiles across roots of two durum wheat genotypes with contrasting rates of Na(+) transport from root to shoot grown in 50 mm NaCl. The Na(+) concentration in Line 149 (low transport genotype) declined across the cortex, being highest in the epidermal and sub-epidermal cells (48 mm) and lowest in the inner cortical cells (22 mm). Na(+) was high in the pericycle (85 mm) and low in the xylem parenchyma (34 mm). The Na(+) profile in Tamaroi (high transport genotype) had a similar trend but with a high concentration (130 mm) in the xylem parenchyma. The K(+) profiles were generally inverse to those of Na(+). Chloride was only detected in the epidermis. These data suggest that the epidermal and cortical cells removed most of the Na(+) and Cl(-) from the transpiration stream before it reached the endodermis, and that the endodermis is not the control point for salt uptake by the plant. The pericycle as well as the xylem parenchyma may be important in the control of net Na(+) loading of the xylem.     

Transport of sodium into the xylem exudate of tobacco

When tobacco (Nicotiana tabacum L. var. Virginia Gold) plants were pretreated with Na (²²Na) several days before detopping, from 2.3 to 4.9% of Na previously accumulated in roots appeared in the xylem exudate in 7 days after detopping. Na from the external medium, however, was readily transported to the exudate. Moreover, the amount of the pretreatment Na that was transported to the exudate was not influenced by the presence of Na in the external medium. When Na was present in the external medium after detopping, about 4% (with an NaNO₃ post treatment) to 10% (with an NaCl post treatment) of the Na transported to the xylem in the 7 days following detopping originated in the vacuoles. Nitrate salts of K or Na in the external medium after detopping resulted in transport of large quantities of the respective cation to the exudate, but not in increased transport of the pretreatment Na. A much larger percentage of the K that was accumulated after detopping than of the Na similarly accumulated was transferred to the xylem exudate.     

Identification and functional characterization of cation-chloride cotransporters in plants

Chloride (Cl(-)) is an essential nutrient and one of the most abundant inorganic anions in plant tissues. We have cloned an Arabidopsis thaliana cDNA encoding for a member of the cation-Cl(-) cotransporter (CCC) family. Deduced plant CCC proteins are highly conserved, and phylogenetic analyses revealed their relationships to the sub-family of animal K(+):Cl(-) cotransporters. In Xenopus laevis oocytes, the A. thaliana CCC protein (At CCC) catalysed the co-ordinated symport of K(+), Na(+) and Cl(-), and this transport activity was inhibited by the 'loop' diuretic bumetanide, a specific inhibitor of vertebrate Na(+):K(+):Cl(-) cotransporters, indicating that At CCC encodes for a bona fide Na(+):K(+):Cl(-) cotransporter. Analysis of At CCC promoter-beta-glucuronidase transgenic Arabidopsis plants revealed preferential expression in the root and shoot vasculature at the xylem/symplast boundary, root tips, trichomes, leaf hydathodes, leaf stipules and anthers. Plants homozygous for two independent T-DNA insertions in the CCC gene exhibited shorter organs such as inflorescence stems, roots, leaves and siliques. The elongation zone of the inflorescence stem of ccc plants often necrosed during bolt emergence, while seed production was strongly impaired. In addition, ccc plants exhibited defective Cl(-) homeostasis under high salinity, as they accumulated higher and lower Cl(-) amounts in shoots and roots, respectively, than the treated wild type, suggesting At CCC involvement in long-distance Cl(-) transport. Compelling evidence is provided on the occurrence of cation-chloride cotransporters in the plant kingdom and their significant role in major plant developmental processes and Cl(-) homeostasis.     

Radial salt transport in corn roots

Primary roots of solution-grown, 5-day-old or 6-day-old seedlings of corn (Zea mays L.) 10 to 14 cm in length were used to study radial salt transport. Measurements were made of the volume of root pressure exudation, salt concentration of the exudate, and rate of salt movement into the xylem exudate. The ³²P uptake, O₂ consumption, and dehydrogenase activity of the root cortex and stele also were studied.

These roots produced copious root pressure exudate containing 4 to 10 times the concentration of ³²P in the external solution. Freshly separated stele from 5-day-old roots accumulated ³²P as rapidly as the cortex from which it was separated and the stele of intact roots also accumulated ³²P. Separated stele has a higher oxygen uptake than cortex. It also shows strong dehydrogenase activity with the tetrazolium test. The high oxygen consumption, ³²P uptake and strong dehydrogenase activity indicate that the cells of the stele probably play a direct role in salt transport.

These data raise doubts concerning theories of radial salt transport into the xylem based on the assumption that the stele is unable to accumulate salt vigorously.

     

Lotus tenuis tolerates the interactive effects of salinity and waterlogging by 'excluding' Na+ and Cl- from the xylem

Salinity and waterlogging interact to reduce growth of poorly adapted species by, amongst other processes, increasing the rate of Na(+) and Cl(-) transport to shoots. Xylem concentrations of these ions were measured in sap collected using xylem-feeding spittlebugs (Philaenus spumarius) from Lotus tenuis and Lotus corniculatus in saline (NaCl) and anoxic (stagnant) treatments. In aerated NaCl solution (200 mM), L. corniculatus had 50% higher Cl(-) concentrations in the xylem and shoot compared with L. tenuis, whereas concentrations of Na(+) and K(+) did not differ between the species. In stagnant-plus-NaCl solution, xylem Cl(-) and Na(+) concentrations of L. corniculatus increased to twice those of L. tenuis. These differences in xylem ion concentrations, which were not caused by variation in transpiration between the two species, contributed to lower net accumulation of Na(+) and Cl(-) in shoots of L. tenuis, indicating that ion transport mechanisms in roots of L. tenuis were contributing to better 'exclusion' of Cl(-) and Na(+) from shoots, compared with L. corniculatus. Root porosity was also higher in L. tenuis, due to constitutive aerenchyma, than in L. corniculatus, suggesting that enhanced root aeration contributed to the maintenance of Na(+) and Cl(-) 'exclusion' in L. tenuis exposed to stagnant-plus-NaCl treatment. Lotus tenuis also had greater dry mass than L. corniculatus after 56 d in NaCl or stagnant-plus-NaCl treatment. Thus, Cl(-) 'exclusion' is a key trait contributing to salt tolerance of L. tenuis, and 'exclusion' of both Cl(-) and Na(+) from the xylem enables L. tenuis to tolerate, better than L. corniculatus, the interactive stresses of salinity and waterlogging.     

Mechanism of sulfate transport inhibition by cycloheximide in plant tissues

Inhibition by cycloheximide of sulfate transport in both barley roots (Hordeum vulgare L.) and potato tuber (Solanum tuberosum L.) increases with increasing inhibitor concentration only to a limited extent, depending on the length of the tissue incubation with the inhibitor. In contrast to this, increasing concentrations of dinitrophenol have a rapid and total inhibitory effect on the active transport. Leucine transport in the same tissues is strongly inhibited by dinitrophenol but is not affected by cycloheximide, whereas incorporation into protein is mainly inhibited by cycloheximide. It appears that the mechanism of transport inhibition by cycloheximide in plant tissues consists in stopping new carrier synthesis and not in the disruption of energy flow. Sulfate carriers show comparable decay rates in barley roots and potato tuber, the mean life being shorter than that of the leucine carriers. These appear more stable in roots than in storage tissues.     

sas1, an Arabidopsis mutant overaccumulating sodium in the shoot, shows deficiency in the control of the root radial transport of sodium

A recessive mutation of Arabidopsis designated sas1 (for sodium overaccumulation in shoot) that was mapped to the bottom of chromosome III resulted in a two- to sevenfold overaccumulation of Na(+) in shoots compared with wild-type plants. sas1 is a pleiotropic mutation that also caused severe growth reduction. The impact of NaCl stress on growth was similar for sas1 and wild-type plants; however, with regard to survival, sas1 plants displayed increased sensitivity to NaCl and LiCl treatments compared with wild-type plants. sas1 mutants overaccumulated Na(+) and its toxic structural analog Li(+), but not K(+), Mg(2)+, or Ca(2)+. Sodium accumulated preferentially over K(+) in a similar manner for sas1 and wild-type plants. Sodium overaccumulation occurred in all of the aerial organs of intact sas1 plants but not in roots. Sodium-treated leaf fragments or calli displayed similar Na(+) accumulation levels for sas1 and wild-type tissues. This suggested that the sas1 mutation impaired Na(+) long-distance transport from roots to shoots. The transpiration stream was similar in sas1 and wild-type plants, whereas the Na(+) concentration in the xylem sap of sas1 plants was 5.5-fold higher than that of wild-type plants. These results suggest that the sas1 mutation disrupts control of the radial transport of Na(+) from the soil solution to the xylem vessels.     

Chloride and sodium influx: a coupled uptake mechanism in the squid giant axon

The squid giant axon was internally dialyzed while the unidirectional fluxes of either Cl or Na were measured. The effects of varying the internal or external concentration of either Na or Cl were studied. Chloride influx was directly proportional to the external Na concentration whereas Cl efflux was unaffected by changes of the external Na concentration between 0 and 425 mM. Neither Cl influx nor efflux were affected by changes of internal Na concentration over the range of 8-158 mM. After ouabain and TTX treatment a portion of the remaining Na influx was directly dependent on the extracellular Cl concentration. Furthermore, when the internal Cl concentration was increased from 0 to 150 mM, the influxes of Cl and Na were decreased by 14 and 11 pmol/cm2.s, respectively. The influx of both ions could be substantially reduced when the axon was depleted of ATP. The influxes of both ions were inhibited by furosemide but unaffected by ouabain. It is concluded that the squid axolemma has an ATP-dependent coupled Na-Cl co-transport uptake mechanism.     

Na^＋ and Water Uptake in Relation to the Radial Reflection Coefficient of Root in Arrowleaf Saltbush Under Salt Stress

The response of halophyte arrowleaf saltbush (Atriplex triangularis Willd) plants to a gradient of salt stress were investigated with hydroponically cultured seedlings. Under salt stress, both the Na+ uptake into root xylem and negative pressures in xylem vessels increased with the elevation of salinity (up to 500 mol/m3) in the root environment. However, the increment in negative pressures in root xylem far from matches the decrease in the osmotic potential of the root bathing solutions, even when the osmotic potential of xylem sap is taken into consideration. The total water potential of xylem sap in arrowleaf saltbush roots was close to the osmotic potential of root bathing solutions when the salt stress was low, but a progressively increased gap between the water potential of xylem sap and the osmotic potential of root bathing solutions was observed when the salinity in the root environment was enhanced. The maximum gap was 1.4 MPa at a salinity level of 500 mol/m3 without apparent dehydration of the tested plants. This discrepancy could not be explained with the current theories in plant physiology. The radial reflection coefficient of root in arrowleaf saltbush decreased with the enhanced salt stress was and accompanied by an increase in the Na+ uptake into xylem sap. However, the relative Na+ in xylem exudates based on the corresponding NaCl concentration in the root bathing solutions showed a tendency of decrease. The results showed that the reduction in the radial reflection coefficient of roots in the arrowleaf saltbush did not lead to a mass influx of NaCl into xylem when the radial reflection coefficient of the root was considerably small; and that arrowleaf saltbush could use small xylem pressures to counterbalance the salt stresses, either with the uptake of large amounts of salt, or with the development of xylem pressures dangerously negative. This strategy could be one of the mechanisms behind the high resistance of arrowleaf saltbush plants to salt stress.     

Apoplastic transport of abscisic acid through roots of maize: effect of the exodermis

The exodermal layers that are formed in maize roots during aeroponic culture were investigated with respect to the radial transport of cis-abscisic acid (ABA). The decrease in root hydraulic conductivity (Lp_r) of aeroponically grown roots was stimulated 1.5-fold by ABA (500 nM), reaching Lp_r values of roots lacking an exodermis. Similar to water, the radial flow of ABA through roots (J_ABA) and ABA uptake into root tissue were reduced by a factor of about three as a result of the existence of an exodermis. Thus, due to the cooperation between water and solute transport the development of the ABA signal in the xylem was not affected. This resulted in unchanged reflection coeffcients for roots grown hydroponically and aeroponically. Despite the well-accepted barrier properties of exodermal layers, it is concluded that the endodermis was the more effective filter for ABA. Owing to concentration polarisation effects, ABA may accumulate in front of the endodermal layer, a process which, for both roots possessing and lacking an exodermis, would tend to increase solvent drag and hence ABA movement into the xylem sap at increased water flow (J_Vr). This may account for the higher ABA concentrations found in the xylem at greater pressure difference. Received: 26 January 1999 / Accepted: 26 May 1999     

Transfer Cells in Roots of Phaseolus coccineus: Ultrastructure and Possible Function in Exclusion of Sodium from the Shoot

A study was made of ultrastructural aspects and ion distributionin roots of Phaseolus coccineus as affected by NaCl and Na₂SO₄salinity. In the proximal region of the root, xylem parenchymacells are differentiated as transfer cells with well developedwall protuberances adjacent to the half-bordered pits of thevessels. The cytoplasm of these transfer cells contains cisternaeof rough endoplasmic reticulum, the number of which was increasedgreatly when the plants were grown in the presence of NaCl orNa₂SO₄. The cisternae of the endoplasmic reticulum are oftenassociated closely with the plasmalemma and interconnected withit by fibrillar bridges. Wall protuberances occur also in the exodermis and epidermisof the more apical region of the root. Their function is stillunknown. P. coccineus excludes Na, but not Cl, from the leaves by retainingit particularly in the proximal region of the root. X-ray microanalysisof unfixed, frozen, hydrated specimens revealed that the transfercell-type xylem parenchyma cells in salt-treated roots accumulatedNa relative to both the adjoining xylem vessels and the corticalcells and showed very high Na/K and Na/Cl ratios. It is suggestedthat the xylem parenchyma cells can reabsorb Na from the vessels,probably in exchange for K, and that Na exclusion from the shootis at least partly mediated by this process. The implicationof this for regulation of salt transport in salt sensitive glycophytesis discussed.     

Waterlogging and salinity effects on two Suaeda salsa populations

Adaptations to combined salinity and waterlogging stress were evaluated in two Suaeda salsa populations from different saline environments. Seedlings were exposed to 1, 200 and 600 mM NaCl in drained or waterlogged sand for 22 days in a glasshouse. Waterlogging did not significantly affect the K(+) /Na(+) ratio or Cl(-) concentration in leaves of either population. Adventitious roots were produced only by the inland population and under the waterlogged condition. X-ray microanalysis showed that S. salsa roots of the intertidal population accumulated more [Na(+) ] and [Cl(-) ] in both the cortex and stele than the roots of the inland population. The ability of roots to exclude Na(+) and Cl(-) was greater in the intertidal population than in the inland population, which may explain why leaves of the intertidal population accumulated less Na(+) and Cl(-) than the leaves of the inland population. The lower level of Cl(-) than Na(+) in leaves of both populations may result from the greater ability of roots to exclude Cl(-) than Na(+) . These traits may help the two S. salsa populations adapt to their different saline environments.     

Effect of lipids on chloride and sodium transport in bean and cotton plants

This paper describes experiments on Cl transport into the roots, stem and leaves of bean plants, the roots of which have been exposed to lipids in the root solution. Monoand digalactose diglyceride strongly increased Cl transport into all plant parts, probably by transport of the glycolipids further into the plant. Phosphatidyl choline increased Cl absorption by the roots, but transport into the stem and leaves was not affected. This phospholipid was only absorbed by the root tissue. ³²P-glycerophosphoryl choline added to the root solution was readily transported and esterified as phospholipid in all plant parts. This chemical did increase Cl uptake by the roots but Cl accumulation in the leaves was reduced by as much as 40%. Phosphatidyl glycerol, phosphatidyl inositol, and sulfolipid increased Cl transport into roots, stem, and leaves, and a high mobility of ³²P-phosphatidyl glycerol was demonstrated. Generally no significant effect of the above lipids on Na transport in beans and cotton was noted except that monogalactose diglyceride did increase Na transport in cotton.     

Na, Cl, and Water Transport by Rat Colon

Segments of the colon of anesthetized rats have been perfused in vivo with isotonic NaCl solutions and isotonic mixtures of NaCl and mannitol. Unidirectional and net fluxes of Na and Cl and the net fluxes of water and mannitol have been measured. Net water transport was found to depend directly on the rate of net Na transport. There was no water absorption from these isotonic solutions in the absence of net solute transport, indicating that water transport in the colon is entirely a passive process. At all NaCl concentrations studied, the lumen was found to be electrically negative to the surface of the colon by 5 to 15 mv. Na fluxes both into and out of the lumen were linear functions of NaCl concentration in the lumen. Net Na absorption from lumen to plasma has been observed to take place against an electrochemical potential gradient indicating that Na is actively transported. This active Na transport has been interpreted in terms of a carrier model system. Cl transport has been found to be due almost entirely to passive diffusion.     

Ion Distribution in Salt-stressed Mature Zea mays Roots in Relation to Ultrastructure and Retention of Sodium

Ultrastructural features and the distribution of soluble ionshave been examined in mature roots of Zea mays plants grownin both NaCl and Na₂SO₄ salinities. When the plants were grown in either salt, the Na concentrationincreased proximally along the root with a concomitant declinein the K concentration. Both trends were reversed in the shoot. X-ray microanalysis of deep-frozen, fully hydrated specimensshowed that in salt-treated roots Na, and Cl, or S were distributedabout stoichiometrically in the cortex and endodermis. Na wasusually less concentrated than the anion in the lumens of thevessels, but was concentrated markedly relative to either Clor S in the adjoining xylem parenchyma cells. In the older, proximal parts of seminal roots of plants grownboth without salt (controls) and in the presence of either NaClor Na₂SO₄, wall developments occurred in xylem parenchyma cellsat the half-bordered pits in which the cell wall became markedlythicker and possessed a loosely packed fibrillar structure.These structures were not comparable with the transfer-celltype of protuberances reported in the roots of other species. In the xylem parenchyma of plants grown in the presence of Na₂SO₄there were dramatic increases in the quantities of rough endoplasmicreticulum, ribosomes, and mitochondria relative both to controlsand NaCl treatments. The results are discussed in relation to the possible functionof the xylem parenchyma of the mature root in the reabsorptionof Na from the xylem sap, which may mitigate adverse effectsof salinity in salt-sensitive glycophytes.     

Efflux of chloride from lactating rat mammary tissue slices

1. The efflux of chloride (using 36Cl) from lactating rat mammary tissue slices has been investigated. 2. Chloride efflux was found to be temperature dependent; lowering the temperature of the incubation medium reduced the fractional efflux. 3. The stilbene derivatives DIDS was without effect on the fractional release of Cl when studied at 20 degrees C. However, DIDS was found to attenuate the increase in efflux found upon transferring the tissue from a medium maintained at 4 degrees C to one at 20 degrees C. 4. The loop-diuretic furosemide, also reduced the temperature-sensitive portion of Cl efflux. 5. Chloride efflux was transiently increased when tissue slices were transferred from a medium containing gluconate as the principal anion to one containing Cl. 6. The results appear to confirm that mammary Cl transport is mediated via anion exchange and via (Na + K + Cl) cotransport.     

Incorporation of 14CO2 in photosynthetic pigments of Chlorella pyrenoidosa

Abscisic acid (ABA) caused a 7–8-fold increase in volume flow in excised bean root systems and this was coupled with an increase in ⁴²K, ³⁶Cl and ²⁴Na flux into the xylem. The transport of ⁴²K and ³⁶Cl increased by a factor larger than the stimulation of volume flow, resulting in an increase in the concentration of those ions in the xylem exudate. Carbonyclcyanide-m-chlorophenyl hydrazone, on the other hand, eliminated ABA-stimulated ⁴²K transport and caused a further inhibition of ⁴²K flux, thus providing additional support for the proposition that ABA stimulation may involve an energised process of ion transport. ABA also increased the accumulation of ²⁴Na and ³⁶Cl in bean root tissue, but not that of ⁴²K.     

Phenotyping and modeling of root hydraulic architecture reveal critical determinants of axial water transport

Water uptake by roots is a key adaptation of plants to aerial life. Water uptake depends on root system architecture (RSA) and tissue hydraulic properties that, together, shape the root hydraulic architecture. This work investigates how the interplay between conductivities along radial (e.g. aquaporins) and axial (e.g. xylem vessels) pathways determines the water transport properties of highly branched RSAs as found in adult Arabidopsis (Arabidopsis thaliana) plants. A hydraulic model named HydroRoot was developed, based on multi-scale tree graph representations of RSAs. Root water flow was measured by the pressure chamber technique after successive cuts of a same root system from the tip toward the base. HydroRoot model inversion in corresponding RSAs allowed us to concomitantly determine radial and axial conductivities, providing evidence that the latter is often overestimated by classical evaluation based on the Hagen–Poiseuille law. Organizing principles of Arabidopsis primary and lateral root growth and branching were determined and used to apply the HydroRoot model to an extended set of simulated RSAs. Sensitivity analyses revealed that water transport can be co-limited by radial and axial conductances throughout the whole RSA. The number of roots that can be sectioned (intercepted) at a given distance from the base was defined as an accessible and informative indicator of RSA. The overall set of experimental and theoretical procedures was applied to plants mutated in ESKIMO1 and previously shown to have xylem collapse. This approach will be instrumental to dissect the root water transport phenotype of plants with intricate alterations in root growth or transport functions.

A model-assisted experimental dissection of architecture and water transport properties of Arabidopsis root systems reveals limiting roles of xylem transport.     

Cadmium distribution in the root tissues of solanaceous plants with contrasting root-to-shoot Cd translocation efficiencies

Root-to-shoot cadmium (Cd) translocation in Solanum torvum is lower than that of the eggplant Solanum melongena; therefore, grafting S. melongena onto S. torvum rootstock can effectively reduce the Cd concentration in eggplant fruits. We hypothesized that Cd transport in S. torvum roots is restricted in the path between the epidermis and xylem vessel; hence, we investigated the Cd distribution in the roots at the micron-scale. Elemental maps of Cd, Zn and Fe accumulation in S. melongena and S. torvum root sections were obtained by synchrotron micro X-ray fluorescence spectrometry. The Cd was localized in both the stele and the epidermis of the S. melongena root cross sections regardless of the distance from the root apex. In S. torvum root sections taken at 30 and 40 mm above the root apex, a higher abundance of Cd was found within the cells of the endodermis and pericycle. The results suggested that the symplastic uptake and xylem loading of Cd in S. torvum roots were restricted, and thereby, the Cd that was unable to be loaded into the xylem accumulated in the endodermis and in the pericycle. Because symplastic uptake differs only slightly between the two species, the difference in xylem loading would explain the comparatively lower Cd concentration in S. torvum shoots.     

Cadmium tolerance in Thlaspi caerulescens: II. Localization of cadmium in Thlaspi caerulescens

Tissue and cellular compartmentation of Cd in roots and leaves of the hyperaccumulator Thlaspi caerulescens was investigated, using energy-dispersive X-ray microanalysis. In roots, Cd was determined in cortex parenchyma cells, endodermis, parenchyma cells of the central cylinder and xylem vessels. In leaves, it was found in cells lying on the way of water migration from the vascular cylinder to epidermal cells, which is in line with passive Cd transport by the transpiration stream. The mechanisms of Cd-detoxification in roots seem to be localized both in apoplast (e.g. binding to cell wall compounds) and inside cells since Cd was accumulated in these two compartments. On the other hand, in leaves Cd was found only in electron-dense deposits inside vacuoles, which suggests that vacuoles are the main compartment of its storage and detoxification in these organs.