A high-density genetic map of hexaploid wheat (Triticum aestivum L.) from the cross Chinese Spring x SQ1 and its use to compare QTLs for grain yield across a range of environments.

A population of 96 doubled haploid lines (DHLs) was prepared from F1 plants of the hexaploid wheat cross Chinese Spring x SQ1 (a high abscisic acid-expressing breeding line) and was mapped with 567 RFLP, AFLP, SSR, morphological and biochemical markers covering all 21 chromosomes, with a total map length of 3,522 cM. Although the map lengths for each genome were very similar, the D genome had only half the markers of the other two genomes. The map was used to identify quantitative trait loci (QTLs) for yield and yield components from a combination of 24 site x treatment x year combinations, including nutrient stress, drought stress and salt stress treatments. Although yield QTLs were widely distributed around the genome, 17 clusters of yield QTLs from five or more trials were identified: two on group 1 chromosomes, one each on group 2 and group 3, five on group 4, four on group 5, one on group 6 and three on group 7. The strongest yield QTL effects were on chromosomes 7AL and 7BL, due mainly to variation in grain numbers per ear. Three of the yield QTL clusters were largely site-specific, while four clusters were largely associated with one or other of the stress treatments. Three of the yield QTL clusters were coincident with the dwarfing gene Rht-B1 on 4BS and with the vernalisation genes Vrn-A1 on 5AL and Vrn-D1 on 5DL. Yields of each DHL were calculated for trial mean yields of 6 g plant(-1) and 2 g plant(-1) (equivalent to about 8 t ha(-1) and 2.5 t ha(-1), respectively), representing optimum and moderately stressed conditions. Analyses of these yield estimates using interval mapping confirmed the group-7 effects on yield and, at 2 g plant(-1), identified two additional major yield QTLs on chromosomes 1D and 5A. Many of the yield QTL clusters corresponded with QTLs already reported in wheat and, on the basis of comparative genetics, also in rice. The implications of these results for improving wheat yield stability are discussed.

The exodermis: a variable apoplastic barrier.

The exodermis (hypodermis with Casparian bands) of plant roots represents a barrier of variable resistance to the radial flow of both water and solutes and may contribute substantially to the overall resistance. The variability is a result largely of changes in structure and anatomy of developing roots. The extent and rate at which apoplastic exodermal barriers (Casparian bands and suberin lamellae) are laid down in radial transverse and tangential walls depends on the response to conditions in a given habitat such as drought, anoxia, salinity, heavy metal or nutrient stresses. As Casparian bands and suberin lamellae form in the exodermis, the permeability to water and solutes is differentially reduced. Apoplastic barriers do not function in an all-or-none fashion. Rather, they exhibit a selectivity pattern which is useful for the plant and provides an adaptive mechanism under given circumstances. This is demonstrated for the apoplastic passage of water which appears to have an unusually high mobility, ions, the apoplastic tracer PTS, and the stress hormone ABA. Results of permeation properties of apoplastic barriers are related to their chemical composition. Depending on the growth regime (e.g. stresses applied) barriers contain aliphatic and aromatic suberin and lignin in different amounts and proportion. It is concluded that, by regulating the extent of apoplastic barriers and their chemical composition, plants can effectively regulate the uptake or loss of water and solutes. Compared with the uptake by root membranes (symplastic and transcellular pathways), which is under metabolic control, this appears to be an additional or compensatory strategy of plants to acquire water and solutes.

Rapid disruption of nitrogen metabolism and nitrate transport in spinach plants deprived of sulphate.

Hydroponically grown spinach plants were deprived of an external source of sulphate after an initial period when the S-supply was sufficient. The time-course of events following this treatment was monitored. The first responses were found in the uptake and translocation of NO(3)(-) and the uptake of SO(4)(2-). The former declined by approximately 50%, the effect being most significant at higher [NO(3)(-)](ext.) while the latter increased 6-fold over a 4 d period. Growth in the absence of external SO(4)(2-) resulted in exhaustion of internal SO(4)(2-) pools, the effect being seen first in roots, then in young leaves and, after a marked delay, in mature leaves. In young leaves, there were dramatic increases in the [NO(3)(-)] and the content of arginine in the first 2 d of S-deprivation. The concentration of glutamine, the most abundant amino acid in S-sufficient conditions, also more than doubled in S-deficient young leaves. The changes in arginine levels were also found in older leaves, but the change in glutamine level was not seen. Assays of nitrate reductase activity (NRA) and nitrate reductase (NR) mRNA from young leaves of S-replete and S-deprived plants revealed a divergence in activity and content only late in the experiments (between days 4 and 8) when results were expressed on a unit leaf basis. However, there were also time-dependent changes in the protein content that kept the specific activities (NRA:protein and RNA:protein) more or less unchanged. The results imply that the impact of S-deficiency on N-utilization are more sensitively monitored by simple measurements of the chemical composition of young leaves than by measurements of NRA or NR transcript abundance. They also suggest that protein synthesis in young leaves is strongly dependent on a continuous supply of SO(4)(2-) from outside the plant.

Root hydraulic conductance: diurnal aquaporin expression and the effects of nutrient stress.

It has been shown that N-, P- and S-deficiencies result in major reductions of root hydraulic conductivity (Lpr) which may lead to lowered stomatal conductance, but the relationship between the two conductance changes is not understood. In a variety of species, Lpr decreases in the early stages of NO3-, H2PO4(2-) and SO4(2-) deprivation. These effects can be reversed in 4-24 h after the deficient nutrient is re-supplied. Diurnal fluctuations of root Lpr have also been found in some species, and an example of this is given for Lotus japonicus. In nutrient-sufficient wheat plants, root Lpr is extremely sensitive to brief treatments with HgCl2; these effects are completely reversible when Hg is removed. The low values of Lpr in N- or P-deprived roots of wheat are not affected by Hg treatments. The properties of plasma membrane (PM) vesicles from wheat roots are also affected by NO3(-)-deprivation of the intact plants. The osmotic permeability of vesicles from N-deprived roots is much lower than that of roots adequately supplied with NO3-, and is insensitive to Hg treatment. In roots of L. japonicus, gene transcripts are found which have a strong homology to those encoding the PIP1 and PIP2 aquaporins of Arabidopsis. There is a very marked diurnal cycle in the abundance of mRNAs of aquaporin gene homologues in roots of L. japonicus. The maxima and minima appear to anticipate the diurnal fluctuations in Lpr by 2-4 h. The temporal similarity between the cycles of the abundance of the mRNAs and root Lpr is most striking. The aquaporin encoded by AtPIP1 is known to have its water permeation blocked by Hg binding. The lack of Hg-sensitivity in roots and PMs from N-deprived roots provides circumstantial evidence that lowered root Lpr may be due to a decrease in either the activity of water channels or their density in the PM. It is concluded that roots are capable, by means completely unknown, of monitoring the nutrient content of the solution in the root apoplasm and of initiating responses that anticipate by hours or days any metabolic disturbances caused by nutrient deficiencies. It is the incoming nutrient supply that is registered as deficient, not the plant's nutrient status. At some point, close to the initiation of these responses, changes in water channel activity may be involved, but the manner in which monitoring of nutrient stress is transduced into an hydraulic response is also unknown.

Effects of abnormal-sterol accumulation on Ustilago maydis plasma membrane H+-ATPase stoichiometry and polypeptide pattern.

Accumulation of 14alpha-methylated sterols or delta8-sterols in Ustilago maydis affected three aspects of the plasma membrane H+-ATPase. Proton transport was reduced in delta8-sterol-accumulating samples, due to an altered H+/ATP stoichiometry. ATP hydrolytic activity was increased, but no direct correlation with the extent or type of abnormal sterol accumulated could be drawn. Finally, Western blot analysis with antibodies against yeast PMA1 revealed a second lighter band (99-kDa band) in all samples from abnormal-sterol-accumulating sporidia. The conclusions are that the 99-kDa band and a reduced stoichiometry are directly linked to the presence of abnormal sterols, while changes in hydrolytic activity are linked only indirectly.

Regulation of expression of a cDNA from barley roots encoding a high affinity sulphate transporter.

A cDNA encoding a high-affinity sulphate transporter has been isolated from barley by complementation of a yeast mutant. The cDNA, designated HVST1, encodes a polypeptide of 660 amino acids (M(r) = 72,550), which is predicted to have 12 membrane-spanning domains and has extensive sequence homology with other identified eukaryotic sulphate transporters. The K(m) for sulphate was 6.9 microM when the HVST1 cDNA was expressed in a yeast mutant deficient in the gene encoding for the yeast SUL1 sulphate transporter. The strong pH-dependency of sulphate uptake when HVST1 was expressed heterologously in yeast suggests that the HVST1 polypeptide is a proton/sulphate co-transporter. The gene encoding HVST1 is expressed specifically in root tissues and the abundance of the mRNA is strongly influenced by sulphur nutrition. During sulphur-starvation of barley, the abundance of mRNA corresponding to HVST1, and the capacity of the roots to take up sulphate, both increase. Upon re-supply of sulphate, the abundance of the mRNA corresponding to HVST1, and the capacity of the roots to take up sulphate, decrease rapidly, concomitant with rises in tissue sulphate, cysteine and glutathione contents. Addition of the cysteine precursor, O-acetylserine, to plants grown with adequate sulphur supply, leads to increases in sulphate transporter mRNA, sulphate uptake rates and tissue contents of glutathione and cysteine. It is suggested, that whilst sulphate, cysteine and glutathione may be candidates for negative metabolic regulators of sulphate transporter gene expression, this regulation may be overridden by O-acetylserine acting as a positive regulator.

SO42- Deprivation Has an Early Effect on the Content of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase and Photosynthesis in Young Leaves of Wheat.

Wheat (Triticum aestivum cv Chinese Spring) supplied with 0.45 mM SO42- for 14 d with relative growth rates (RGR) of 0.22 to 0.24 d-1 was deprived of S for 7 to 8 d. There was no significant effect on RGR or leaf development (leaf 2 length was constant; leaf 3 expanded for 2-4 d; leaf 4 emerged and elongated throughout the experiment) during the S deprivation. In controls the net assimilation rate (A) closely reflected leaf ontogeny. S deprivation affected A in all leaves, particularly leaf 4, in which A remained at 8 to 10 [mu]mol CO2 m-2 s-1, whereas in controls A rose steadily to >20 [mu]mol CO2 m-2 s-1. In leaf 2, with a fully assembled photosynthetic system, A decreased in S-deprived plants relative to controls only at the end of the experiment. Effects on A were not due to altered stomatal conductance or leaf internal [CO2] ([C]i); decreases in the initial slope of A/[C]i curves indicated an effect of S deprivation on the carboxylase efficiency. Measurement of Rubisco activity and large subunit protein abundance paralleled effects on A and A/[C]i in S-deprived leaves. Negative effects on photosynthesis in S-deprived plants are discussed in relation to mobilization of S reserves, including Rubisco, emphasizing the need for continuous S supply during vegetative growth.

Molecular cloning and characterisation of asparagine synthetase from Lotus japonicus: dynamics of asparagine synthesis in N-sufficient conditions.

Two cDNA clones, LJAS1 and LJAS2, encoding different asparagine synthetases (AS) have been identified and sequenced and their expression in Lotus japonicus characterised. Analysis of predicted amino acid sequences indicted a high level of identity with other plant AS sequences. No other AS genes were detected in the L. japonicus genome. LJAS1 gene expression was found to be root-enhanced and lower levels of transcript were also identified in photosynthetic tissues. In contrast, LJAS2 gene expression was root-specific. These patterns of AS gene expression are different from those seen in pea. AS gene expression was monitored throughout a 16 h light/8 h dark day, under nitrate-sufficient conditions. Neither transcript showed the dark-enhanced accumulation patterns previously reported for other plant AS genes. To evaluate AS activity, the molecular dynamics of asparagine synthesis were examined in vivo using 15N-ammonium labelling. A constant rate of asparagine synthesis in the roots was observed. Asparagine was the most predominant amino-component of the xylem sap and became labelled at a slightly slower rate than the asparagine in the roots, indicating that most root asparagine was located in a cytoplasmic 'transport' pool rather than in a vacuolar 'storage' pool. The steady-state mRNA levels and the 15N-labelling data suggest that light regulation of AS gene expression is not a factor controlling N-assimilation in L. japonicus roots during stable growth in N-sufficient conditions.

Plant members of a family of sulfate transporters reveal functional subtypes.

Three plant sulfate transporter cDNAs have been isolated by complementation of a yeast mutant with a cDNA library derived from the tropical forage legume Stylosanthes hamata. Two of these cDNAs, shst1 and shst2, encode high-affinity H+/sulfate cotransporters that mediate the uptake of sulfate by plant roots from low concentrations of sulfate in the soil solution. The third, shst3, represents a different subtype encoding a lower affinity H+/sulfate cotransporter, which may be involved in the internal transport of sulfate between cellular or subcellular compartments within the plant. The steady-state level of mRNA corresponding to both subtypes is subject to regulation by signals that ultimately respond to the external sulfate supply. These cDNAs represent the identification of plant members of a family of related sulfate transporter proteins whose sequences exhibit significant amino acid conservation in filamentous fungi, yeast, plants, and mammals.

Isolation of a cDNA from Saccharomyces cerevisiae that encodes a high affinity sulphate transporter at the plasma membrane.

Resistance to selenate and chromate, toxic analogues of sulphate, was used to isolate a mutant of Saccharomyces cerevisiae deficient in the capacity to transport sulphate into the cells. A clone which complements this mutation was isolated from a cDNA library prepared from S. cerevisiae poly(A)+ RNA. This clone contains an insert which is 2775 bp in length and has a single open reading frame that encodes a 859 amino acid polypeptide with a molecular mass of 96 kDa. Sequence motifs within the deduced amino acid sequence of this cDNA (SUL1) show homology with conserved areas of sulphate transport proteins from other organisms. Sequence analysis predicts the position of 12 putative membrane spanning domains in SUL1. When the cDNA for SUL1 was expressed in S. cerevisiae, a high affinity sulphate uptake activity (Km = 7.5 +/- 0.6 microM for SO2-4) was observed. A genomic mutant of S. cerevisiae in which 1096 bp were deleted from the SUL1 coding region was constructed. This mutant was unable to grow on media containing less than 5 mM sulphate unless complemented with a plasmid containing the SUL1 cDNA. We conclude that the SUL1 cDNA encodes a S. cerevisiae high affinity sulphate transporter that is responsible for the transfer of sulphate across the plasma membrane from the external medium.

Ca(2+)-ATPase-driven calcium accumulation in Ustilago maydis plasma membrane vesicles.

Ca2+ transport has been measured across plasma membrane vesicles isolated from cells of Ustilago maydis. This transport was found to be ATP- (or to a lesser extent GTP) and Mg(2+)-dependent. Inconsistent release of Ca2+ from intact vesicles was obtained using the calcium ionophore A23187. However, Ca2+ was released by Triton X-100 in a concentration-dependent manner. Transport was inhibited by vanadate (> 50%) and erythrosin B (about 50%), I50 being about 10 microM for both inhibitors. In the presence of the protonophores CCCP or gramicidin, partial inhibition of Ca2+ transport (about 20%) was observed, but the Ca(2+)-channel blockers, nifedipine, diltiazem and verapamil had no effect, although the latter inhibited proton transport. The results indicate that Ca2+ transport in U. maydis is regulated by a P-type ATPase with similar properties to that found in higher plants.

Lipid composition and proton transport in Penicillium cyclopium and Ustilago maydis plasma membrane vesicles isolated by two-phase partitioning.

Plasma membranes have been isolated and purified from two species of fungi, Penicillium cyclopium and Ustilago maydis, using a two-phase aqueous polymer technique. The membranes were characterised using marker enzyme assays (e.g., vanadate-sensitive (Mg(2+)-K+)-ATPase and glucan synthetase II) and lipid composition (sterol enrichment, increased phosphatidylethanolamine/phosphatidylcholine ratio, and the absence of diphosphatidylglycerol). The proton-pumping activities of the plasma membrane-bound H(+)-ATPases from these species were compared. H(+)-ATPase activity was found to be greater in U. maydis than in P. cyclopium, which was attributed to differences in orientation of the plasma membrane vesicles. There was evidence to suggest the presence of redox chain activity in the plasma membranes of both species.

Contrasting responses of sulphate and phosphate transport in barley (Hordeum vulgare L.) roots to protein-modifying reagents and inhibition of protein synthesis.

The uptake of sulphate into roots of barley seedlings is highly sensitive to phenylglyoxal (PhG), an arginine-binding reagent. Uptake was inhibited by >80% by a 1-h pre-treatment of roots with 0.45 mol · m(-3) PhG. Inhibition was maximal in pre-treatment solutions buffered between pH 4.5 and 6.5. Phosphate uptake, measured simultaneously by double-labelling uptake solutions with (32)P and (35)S, was less susceptible to inhibition by PhG, particularly at pH <6.5, and was completely insensitive to the less permeant reagent p-hydroxyphenylglyoxal (OH-PhG) administered at 1 mol · m(-3) at pH at 5.0 or 8.2; sulphate uptake was inhibited in -S plants by 90% by OH-PhG-treatment. Root respiration in young root segments was unaffected by OH-PhG pre-treatment for 1 h and inhibited by only 17% after 90 min pre-treatment. The uptake of both ions was inhibited by the dithiol-specific reagent, phenylarsine oxide even after short exposures (0.5-5.0 min). Sulphate uptake was more severely inhibited than that of phosphate, but in both cases inhibition could be substantially reversed by 5 min washing of treated roots by 5 mol · m(-3) dithioerythritol. After longer pre-treatment (50 min) with phenylarsine oxide, inhibition of the ion fluxes was not relieved by washing with dithioerythritol. Inhibition of sulphate influx by PhG was completely reversed by washing the roots for 24 h with culture solution lacking the inhibitor. The reversal was dependent on protein synthesis; less than 20% recovery was seen in the presence of 50 mmol · m(-3) cycloheximide. Sulphate uptake declined rapidly when -S roots were treated with cycloheximide. In the same roots the phosphate influx was little affected, small significant inhibitions being seen only after 4 h of treatment. Respiration was depressed by only 20% in apical and by 31% in basal root segments by cycloheximide pre-treatment for 2 h. Similar rates of collapse of the sulphate uptake and insensitivity of phosphate uptake were seen when protein synthesis was inhibited by azetidine carboxylic acid, p-fluorophenylalanine and puromycin. Considering the effects of all of the protein-synthesis inhibitors together leads to the conclusion that the sulphate transporter itself, or some essential sub-component of the uptake system, turns over rapidly with a half-time of about 2.5 h. The turnover of the phosphate transporter is evidently much slower. The results are discussed in relation to strategies for identifying the transport proteins and to the regulation of transporter activity during nutrient stress.

Sulphate deprivation depresses the transport of nitrogen to the xylem and the hydraulic conductivity of barley (Hordeum vulgare L.) roots.

During the first 4 d after the removal of SO 4 (2-) from cultures of young barley plants, the net uptake of (15)N-nitrate and the transport of labelled N to the shoot both decline. This occurred during a period in which there was no measurable change in plant growth rate and where the incorporation of [(3)H]leucine into membrane and soluble proteins was unaffected. Reduced N translocation was associated with six- to eightfold increases in the level of asparagine and two- to fourfold increases in glutamine in root tissue; during the first 4 d of SO 4 (2-) deprivation there were no corresponding increases in amides in leaf tissue. The provision of 1 mol · m(-3) methionine halted, and to some extent reversed the decline in NO 3 (-) uptake and N translocation which occurred during continued SO 4 (2-) deprivation. This treatment had relatively little effect in lowering amide levels in roots. Experiments with excised root systems indicated that SO 4 (2-) deprivation progressively lowered the hydraulic conductivity, Lp, of roots; after 4 d the Lp of SO 4 (2-) -deprived excised roots was only 20% of that of +S controls. In the expanding leaves of intact plants, SO 4 (2-) deprivation for 5 d was found to lower stomatal conductance, transpiration and photosynthesis, in the order given, to 33%, 37% and 18% of control values. The accumulation of amides in roots is probably explained by a failure to export either the products of root nitrate assimilation or phloem-delivered amino-N. This may be correlated with the lowered hydraulic conductivity. Enhanced glutamine and-or asparagine levels probably repressed net uptake of NO 3 (-) and (13)NO 3 (-) influx reported earlier (Clarkson et al. 1989, J. Exp. Bot. 40, 953-963). Attention is drawn to the similar hydraulic signals occurring in the early stages of several different types of mineral-nutrient stresses.

Sulphate influx in wheat and barley roots becomes more sensitive to specific protein-binding reagents when plants are sulphate-deficient.

When young wheat (Triticum aestivum L.) or barley (Hordeum vulgare L.) plants were deprived of an external sulphate supply (-S plants), the capacity of their roots to absorb sulphate, but not phosphate or potassium, increased rapidly (derepression) so that after 3-5 d it was more than tenfold that of sulphate-sufficient plants (+S plants). This increased capacity was lost rapidly (repression) over a 24-h period when the sulphate supply was restored. There was little effect on the uptake of L-methionine during de-repression of the sulphate-transport system, but S input from methionine during a 24-h pretreatment repressed sulphate influx in both+S and-S plants.Sulphate influx of both+S and-S plants was inhibited by pretreating roots for 1 h with 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) at concentrations > 0.1 mol · m(-3). This inhibition was substantially reversed by washing for 1 h in DIDS-free medium before measuring influx. Longer-term pretreatment of roots with 0.1 mol·m(-3) DIDS delayed de-repression of the sulphatetransport system in-S plants but had no influence on+S plants in 3 d.The sulphydryl-binding reagent, n-ethylmaleimide, was a very potent inhibitor of sulphate influx in-S roots, but was much less inhibitory in +S roots. Its effects were essentially irreversible and were proportionately the same at all sulphate concentrations within the range of operation of the high-affinity sulphate-transport system. Inhibition of influx was 85-96% by 300 s pretreatment by 0.3 mol·m(-3) n-ethylmaleimide. No protection of the transport system could be observed by including up to 50 mol·m(-3) sulphate in the n-ethylmaleimide pre-treatment solution. A similar differential sensitivity of-S and+S plants was seen with p-chloromercuriphenyl sulphonic acid.The arginyl-binding reagent, phenylglyoxal, supplied to roots at 0.25 or 1 mol·m(-3) strongly inhibited influx in-S wheat plants (by up to 95%) but reduced influx by only one-half in+S plants. The inhibition of sulphate influx in-S plants was much greater than that of phosphate influx and could not be prevented by relatively high (100 mol·m(-3) sulphate concentrations accompanying phenylglyoxal treatment. Effects of phenylglyoxal pretreatment were unchanged for at least 30 min after its removal from the solution but thereafter the capacity for sulphate influx was restored. The amount of 'new' carrier appearing in-S roots was far greater than in+S roots over a 24-h period.The results indicate that, in the de-repressed state, the sulphate transporter is more sensitive to reagents binding sulphydryl and arginyl residues. This suggests a number of strategies for identifying the proteins involved in sulphate transport.

Temperature dependent factors influencing nutrient uptake: an analysis of responses at different levels of organization.

Ion fluxes show a characteristically biochemical dependence on temperature when observed at the membrane level and over short periods after a perturbation of temperature. The primary active transport systems are enzymic and are dependent both on substrate supply and on changes in protein conformation. The hydrophobic parts of the proteins are surrounded by lipid molecules whose physical state may crucially affect conformation changes. These lipids may undergo transitions from a fluid to a gel state at temperatures occurring in the natural environment. It will be noted that the concepts developed in model systems of pure phospholipid/protein interactions cannot be very readily applied to the spatially heterogeneous assemblies of lipid molecules and transport proteins in real cell membranes. While it is obvious that ion transport rates are responsive to temperature changes in a given cell, it is difficult to explain exactly which components of the transport process become limiting. We will show that, on cooling, the membrane potential can initially be greatly disturbed when temperature is changed and that this may be related to ATP supply to H+-translocating ATPase. This affects the driving force for all other solutes. When temperature is lowered the permeability coefficients for most ions are reduced and yet it is commonly found that diffusive efflux of ions increases in the cold. We attempt to explain this paradox on the basis of driving forces and metabolic regulation of ion transport. Acclimatory changes occur on extended exposure of a cell or an organism to a reduced growth temperature. Some of these changes occur at the membrane level and relate to lipid composition and modulation of carrier activity. Others involve changes in the relative size and sometimes the morphology of the root system. We will show that these processes lessen the temperature dependence of ion transport and ensure that the intake of nutrients does not limit growth at low temperatures. These acclimatory changes are seen as part of the general process of regulation of nutrient uptake.

Effect of nitrogen stress and abscisic acid on nitrate absorption and transport in barley and tomato.

The potential of barley (Hordeum vulgare L.) and tomato (Lycopersicon esculentum Mill.) roots for net NO 3 (-) absorption increased two-to five fold within 2 d of being deprived of NO 3 (-) supply. Nitrogen-starved barley roots continued to maintain a high potential for NO 3 (-) absorption, whereas NO 3 (-) absorption by tomato roots declined below control levels after 10 d of N starvation. When placed in a 0.2 mM NO 3 (-) solution, roots of both species transported more NO 3 (-) and total solutes to the xylem after 2 d of N starvation than did N-sufficient controls. However, replenishment of root NO 3 (-) stores took precedence over NO 3 (-) transport to the xylem. Consequently, as N stress became more severe, transport of NO 3 (-) and total solutes to the xylem declined, relative to controls. Nitrogen stress caused an increase in hydraulic conductance (L p) and exudate volume (J v) in barley but decrased these parameters in tomato. Nitrogen stress had no significant effect upon abscisic acid (ABA) levels in roots of barley or flacca (a low-ABA mutant) tomato, but prevented an agerelated decline in ABA in wild-type tomato roots. Applied ABA had the same effect upon barley and upon the wild type and flacca tomatoes: L p and J v were increased, but NO 3 (-) absorption and NO 3 (-) flux to the xylem were either unaffected or sometimes inhibited. We conclude that ABA is not directly involved in the normal changes in NO 3 (-) absorption and transport that occur with N stress in barley and tomato, because (1) the root ABA level was either unaffected by N stress (barley and flacca tomato) or changed, after the greatest changes in NO 3 (-) absorption and transport and L p had been observed (wild-type tomato); (2) changes in NO 3 (-) absorption/transport characteristics either did not respond to applied ABA, or, if they did, they changed in the direction opposite to that predicted from changes in root ABA with N stress; and (3) the flacca tomato (which produces very little ABA in response to N stress) responded to N stress with very similar changes in NO 3 (-) transport to those observed in the wild type.

Growth response of barley and tomato to nitrogen stress and its control by abscisic acid, water relations and photosynthesis.

Barley (Hordeum vulgare L.) and tomato Lycopersicon esculentum Mill.) were grown hydroponically and examined 2, 5, and 10 d after being deprived of nitrogen (N) supply. Leaf elongation rate declined in both species in response to N stress before there was any reduction in rate of dryweight accumulation. Changes in water transport to the shoot could not explain reduced leaf elongation in tomato because leaf water content and water potential were unaffected by N stress at the time leaf elongation began to decline. Tomato maintained its shoot water status in N-stressed plants, despite reduced water absorption per gram root, because the decline in root hydraulic conductance with N stress was matched by a decline in stomatal conductance. In barley the decline in leaf elongation coincided with a small (8%) decline in water content per unit area of young leaves; this decline occurred because root hydraulic conductance was reduced more strongly by N stress than was stomatal conductance. Nitrogen stress caused a rapid decline in tissue NO 3 (-) pools and in NO 3 (-) flux to the xylem, particularly in tomato which had smaller tissue NO 3 (-) reserves. Even in barley, tissue NO 3 (-) reserves were too small and were mobilized too slowly (60% in 2 d) to support maximal growth for more than a few hours. Organic N mobilized from old leaves provided an additional N source to support continued growth of N-stressed plants. Abscisic acid (ABA) levels increased in leaves of both species within 2 d in response to N stress. Addition of ABA to roots caused an increase in volume of xylem exudate but had no effect upon NO 3 (-) flux to the xylem. After leaf-elongation rate had been reduced by N stress, photosynthesis declined in both barley and tomato. This decline was associated with increased leaf ABA content, reduced stomatal conductance and a decrease in organic N content. We suggest that N stress reduces growth by several mechanisms operating on different time scales: (1) increased leaf ABA content causing reduced cell-wall extensibility and leaf elongation and (2) a more gradual decline in photosynthesis caused by ABA-induced stomatal closure and by a decrease in leaf organic N.

The regulation of phosphatidylcholine biosynthesis in rye (Secale cereale) roots. Stimulation of the nucleotide pathway by low temperature.

The incorporation of [14C]choline chloride and [14C]glycerol into segments taken from rye (Secale cereale L., cv. Rheidal) roots was greater in segments from roots grown at 5 degrees C than in segments taken from roots growing at 20 degrees C. The incorporation was measured at the temperature at which the root had been growing. Measurements in vitro of the enzymes of the nucleotide pathway showed activity of choline kinase (EC 2.7.1.32), choline-phosphate cytidylyltransferase (EC 2.7.7.15) and cholinephosphotransferase (EC 2.7.8.2) to be higher in homogenates from the cooler roots when assayed at 5 degrees C than the activities assayed at 20 degrees C in the 20 degrees C-root homogenates. Changes in vivo in the pool sizes of the CDP-base intermediates with temperature, relative differences in nucleotide-pathway-enzyme activities and a pulse-chase experiment with [14C]choline indicated that the rate-limiting step for phosphatidylcholine biosynthesis in this tissue, at both temperatures, was the reaction catalysed by cytidylyltransferase.

Acclimation of potassium influx in rye (Secale cereale) to low root temperatures.

The influx of K(+)((86)Rb(+)) into intact roots of rye (Secale cereale L. cv. Rheidal) exposed to a differential temperature (DT) between the root (8° C) and shoot (20° C) is initially reduced compared with warm-grown (WG) controls with both shoot and root maintained at 20° C. Over a period of 3 d, however, K(+)-influx rates into DT plants are restored to levels similar to or greater than those of the WG controls, the absolute rates of K(+) influx being strongly dependent upon the shoot/root ratio. Acclimation in DT plants results in a reduction of K(+) influx into the apical (0-2 cm) region of the seminal root which is associated with a compensatory increase in K(+) influx into the more mature, basal regions of the root. Values of V max and apparent K m for K(+) influx into DT plants were similar to those for WG plants at assay temperatures of 8° C and 20° C except for an increase in the apparent K m at 8° C. The influx of K(+) from solutions containing 0.6 mol·m(-3) K(+) into both WG and DT plants was found to be linearly related to assay temperature over the range 2-27° C, and the temperature sensitivity of K(+) influx to be dependent upon shoot/root ratio. At high shoot/root ratios, the ratio of K(+) influx at 20° C:K(+) influx at 8° C for WG plants approached a minimum value of 1.9 whereas that for DT plants approached unity indicating that K(+) influx into DT plants has a large temperature-insensitive component. Additionally, when plants were grown in solutions of low potassium concentration, K(+) influx into DT plants was consistently greater than that into WG plants, in spite of having a greater root potassium concentration ([K(+)]int). This result indicates some change in the regulation of K(+) influx by [K(+)]int in plants exposed to low root temperatures. We suggest that K(+) influx into rye seedlings exposed to low root temperatures is regulated by the increased demand placed on the root system by a proportionally larger shoot and that the acclimation of K(+) influx to low temperatures may be the result of an increased hydraulic conductivity of the root system.