David T. Clarkson

Nitrate and ammonium nutrition of plants : physiological and molecular perspectives.

Nitrogen is the mineral nutrient that plants need in the greatest quantities and the one that most frequently limits plant growth and crop yields. Most plants get their nitrogen (N) from the soil as either nitrate or ammonium, with some species showing a strong preference for one ionic form over the other. The uptake of nitrate and ammonium ions by roots involves a complex set of membrane transport systems that includes both high- and low-affinity transporters; net uptake rates can also be strongly influenced by the rate at which these ions efflux from root cells. Here we review our current picture of the mechanisms responsible for the uptake and efflux of nitrate and ammonium, attempting to integrate the large body of physiological data with the recent advances in the molecular biology of nitrate and ammonium transporters in bacteria and algae as well as in higher plants. We also review what is known at the physiological and molecular levels about the regulation of the N uptake systems, a process which involves both positive signals from soil nitrate or ammonium and feedback inhibitory signals that are generated by the plants internal N status

Diurnal variations in hydraulic conductivity and root pressure can be correlated with the expression of putative aquaporins in the roots of Lotus japonicus

Abstract. The hydraulic conductivity of excised roots (Lpr) of the legume Lotus japonicus (Regel) K. Larsen grown in mist (aeroponic) and sand cultures, was found to vary over a 5-fold range during a day/night cycle. This behaviour was seen when Lpr was measured in roots exuding, either under root pressure (osmotic driving force), or under an applied hydrostatic pressure of 0.4 MPa which produced a rate of water flow similar to that in a transpiring plant. A similar daily pattern of variation was seen in plants grown in natural daylight or in controlled-environment rooms, in plants transpiring at ambient rates or at greatly reduced rates, and in plants grown in either aeroponic or sand culture. When detached root systems were connected to a root pressure probe, a marked diurnal variation was seen in the root pressure generated. After excision, this circadian rhythm continued for some days. The hydraulic conductivity of the plasma membrane of individual root cells was measured during the diurnal cycle using a cell pressure probe. Measurements were made on the first four cell layers of the cortex, but no evidence of any diurnal fluctuation could be found. It was concluded that the conductance of membranes of endodermal and stelar cells may be responsible for the observed diurnal rhythm in root Lpr. When mRNAs from roots were probed with cDNA from the Arabidopsis aquaporin AthPIP1a gene, an abundant transcript was found to vary in abundance diurnally under high-stringency conditions. The pattern of fluctuations resembled closely the diurnal pattern of variation in root Lpr. The plasma membranes of root cells were found to contain an abundant hydrophobic protein with a molecular weight of about 31 kDa which cross-reacted strongly to an antibody raised against the evolutionarily conserved N-terminal amino acid sequence of AthPIP1a.

Responses of wheat plants to nutrient deprivation may involve the regulation of water-channel function

The sap flow (Jv) and the osmotic hydraulic conductance (L0) of detached, exuding root systems from wheat (Triticum aestivum L. cv. Chinese Spring) plants deprived of nitrogen for 5 d (— N) or of phosphorus for 7 d (—P), were measured and compared with controls receiving a complete nutrient supply. In the roots of — N and — P plants, Jv and L0 decreased markedly, but between 4 and 24 h after resupplying N to — N plants (NRS plants) and P to — P plants (PRS plants), Jv and Lo recovered to values similar to those of control plants. Values of Jv and L0 were always greater during the light period than during the dark, due to the diurnal variation of these parameters. Reducing transpiration in the light had no effect on Jv and L0 of — N and — P plants. Sap flow and L0 were also determined using individual axes from plants which had been grown with their roots divided between nutrient-deficient (- N or- P) solution and a complete nutrient solution. Differences were observed in Jv and L0 between axes of the same plant, but stomatal conductance (Gs), which was also measured, was not affected in these split-root experiments. In control plants, Jv and L0 declined sharply to values similar to those of roots from — N and — P plants after HgCl2 treatment (50 μM), but were restored by treating with 5 mM dithiothreitol. In plasma membranes from — N and — P roots, the amount of stigmasterol increased relative to sitosterol compared with control roots. The degree of unsaturation of bound fatty acids also increased, compared with controls, as a result of a decline in the relative amounts of 16∶0 and 18∶0 and an increase in 18∶2. Plasma-membrane fluidity, estimated by steady-state fluorescence polarisation using 1,6-diphenyl hexatriene, showed that the plasma membranes from nutrient-deprived plants were less fluid than those from control plants, measured during both the light and dark periods and in split-root experiments. In NRS plants, the relative abundance of sitosterol increased, so that the stigmasterol/sitosterol ratio returned to a value similar to that of controls. However, in PRS plants, the difference in stigmasterol/sitosterol ratio was maintained, compared with controls. The degree of unsaturation of bound fatty acids, membrane fluidity and the hydraulic conductivity of root systems also recovered in NRS and PRS plants to values similar to those of control plant plasma membranes. The results obtained suggested that — N and — P treatment decreased L0, by reducing either the activity or the abundance of Hg-sensitive water channels. Also, there may be an interaction between the increase in membrane lipid ordering and the decrease in L0.

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 NO3-pools and in NO3-flux to the xylem, particularly in tomato which had smaller tissue NO3-reserves. Even in barley, tissue NO3-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 NO3-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.

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 by 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 μM for SO42−) was observed. A genomic mutant of S. cerevisiae in which 1096 by 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.

Relationships between structural development and the absorption of ions by the root system of Cucurbita pepo.

SummaryIn both the seminal axis and lateral roots of Cucurbita pepo L. the formation of large central xylem elements and the commencement of secondary cambial activity occur 10–20 cm from the root tip. Concomitant with or slightly preceding these developments there are changes in the structure of the walls of endodermal cells where the lignified casparian band spreads along the radial wall and substances staining with Sudan IV are deposited in both radial and tangential walls. At distances more than 30 cm from the tip of primary roots the radius of the stele increases considerably causing splits in the cortex. The endodermis is stretched and the suberin becomes organized in a lamellar form.Against this background of anatomical change certain of the transport capabilities of the root are retained while others are lost. Using an apparatus for measuring the uptake of tracers by segments of intact roots it was found that neither the uptake nor translocation of potassium seem to be affected by the suberization of the endodermis or by secondary thickening, while the translocation of calcium is virtually eliminated when these processes begin. As the root ages its ability to absorb phosphate declines although the translocation of the phosphate absorbed is much less affected by structural development than that of calcium.The observed rates of potassium uptake by complete root systems could be predicted quite accurately from the average of segment uptake data suggesting that the method used gives reliable results.

Mineral Nutrition: Divalent Cations, Transport and Compartmentation

“Heavy metals” and “trace elements” were last reviewed in this series by Lonera-gan (1982) and Fe was also covered in Luttge and Clarkson (1985). The physiology and ecophysiology of “heavy metal plants” is discussed by Ernst (1982). In this review we will discuss the membrane transport and internal compartmentation of divalent cations in the light of what has been discovered about calcium. We do this without exhaustive reference to the frequently reviewed calcium literature (Evans 1988). The divalent cations encountered by plants make up a diverse group of elements some of which have major nutrient functions, others have well-recognized roles as trace elements, others such as nickel and cobalt are suspected trace elements while others, frequently concentrated by the activities of man, are highly toxic to most plants. To introduce some cohesion into the review, we have concentrated on possible ways in which their activities in the cytoplasm might be controlled. We have excluded Fe2+ from this review because of the specialized nature of its absorption by roots.

Influence of phosphate-stress on phosphate absorption and translocation by various parts of the root system of Hordeum vulgare L. (barley).

Plants of Hordeum vulgare (barley) were grown initially in a solution containing 150 μM phosphate and then transferred on day 6 to solutions with (+P) and without (-P) phosphate supplied. After various times plants from these treatments were supplied with labelled phosphate. Analysis of plant growth and rates of labelled phosphate uptake showed that a general enhancement of uptake and translocation was found, in plants which had been in the-P solution, several days before the rate of dry matter accumulation was affected. Subsequently a detailed analysis of phosphate uptake by segments of intact root axes showed that the enhancement of phosphate uptake by P-stress occurred first in the old and mature parts of the seminal root axis and last in the young zones 1 cm from the root apex. During this transition period there were profound changes in the pattern of P absorption along the length of the root. Most of the additional P absorbed in response to P-stress was translocated to the shoot, particularly in older zones of the axis. Enhancement of phosphate uptake in young zones of nodal axes occurred at an earlier stage than in seminal axes. The results are related to the P-status of shoots and root zones and discussed in relation to the general control by the shoot of phosphate transport in the root.

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 SO42-from cultures of young barley plants, the net uptake of 15N-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 [3H]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 SO42-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 NO3-uptake and N translocation which occurred during continued SO42-deprivation. This treatment had relatively little effect in lowering amide levels in roots. Experiments with excised root systems indicated that SO42-deprivation progressively lowered the hydraulic conductivity, Lp, of roots; after 4 d the Lp of SO42--deprived excised roots was only 20% of that of +S controls. In the expanding leaves of intact plants, SO42-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 NO3-and 13NO3-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.

Mineral Nutrition: Inducible and Repressible Nutrient Transport Systems

Compartmentation and transport of solutes across membranes of plants, including movements of nutrient anions and cations, are governed by primary active H+-pumping, predominantly by H+-ATPases at the plasmalemma and tonoplast. Nutrient transports are linked to the H+-pumps via cotransport, antiport, and uniport mechanisms. Elucidation of the physiological, biochemical, and genetic regulation of the primary active H+-pumps is making rapid progress, and complex networks of control are revealed. Thus, the catalytic mechanism of the plasmalemma ATPase requires its phosphorylation at one site (Briskin and Leonard 1982; Briskin and Poole 1983a, b; Briskin 1986,1988), but the enzyme also needs phosphorylation at another site for its activation. Various systems like zucchini hypocotyls (Salimath and Marme 1983), corn coleoptiles (Veluthambi and Poovaiah 1984), spinach leaves (Aducci et al. 1987) and apple fhiit membranes (Battey and Venis 1988) were shown to have Ca2+ and calmodulin-regulated, membrane-related protein kinases. In corn root cells both the plasmalemma and the tonoplast contain protein kinase activities (Ladror and Zielinski 1989). Calcium was found to regulate the plasmalemma H+-pump of Neurospora crassa (Lew 1989). From pea plants a calcium/calmodulin-reg- ulated protein kinase was isolated (Blowers et al. 1985), and in oat roots the plasmalemma H+-ATPase was demonstrated to be the main substrate of the membrane-bound and calcium/calmodulin-regulated protein kinase (Schaller and Sussman 1987, 1988). Cytosolic calcium itself inter alia is controlled by a Ca2+-extruding ATPase at the plasmalemma, which also appears to be calmodulin-dependent in leaves of maize (Robinson et al. 1988) but not of Commelina communis (Graf and Weiler 1989). Lipid interactions are involved. A newly discovered plant phospholipid similar to the platelet-activating factor of animals stimulates the protein-kinase and H+-ATPase in plant-membranes vesicles of plasmalemma and tonoplast origin (Scherer and Stoffel 1987; Scherer et al. 1988). The phosphatidylinositol cycle known for the calcium-messenger and protein-phospho- rylation mechanisms of animals is also operative in plants (Poovaiah et al. 1987; Owen 1988) and is associated with the plasmalemma (Pfaffman et al. 1987; Wheeler and Boss 1987; Morse et al. 1989). Sequencing and cloning of polypeptide-subunits of the H+- pumps is advancing fast (Bowman et al. 1988a, b; Manolson et al. 1988; Zimniak et al. 1988; Nelson and Nelson 1988; Nelson et al. 1989; Serrano 1989, 1990), and this offers insights into regulation at the genetic level.