The hydraulic conductivity of excised roots (Lp(r)) 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 Lp(r) 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 Lp(r). 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 Lp(r). 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.

A mathematical model is presented that describes permeation of hydrogen peroxide across a cell membrane and the implications of solute decomposition by catalase inside the cell. The model was checked and analysed by means of a numerical calculation that raised predictions for measured osmotic pressure relaxation curves. Predictions were tested with isolated internodal cells of CHARA: corallina, a model system for investigating interactions between water and solute transport in plant cells. Series of biphasic osmotic pressure relaxation curves with different concentrations of H(2)O(2) of up to 350 mol m(-3) are presented. A detailed description of determination of permeability (P(s)) and reflection coefficients (sigma(s)) for H(2)O(2) is given in the presence of the chemical reaction in the cell. Mean values were P(s)=(3.6+/-1.0) 10(-6) m s(-1) and sigma(s)=(0.33+/-0.12) (+/-SD, N=6 cells). Besides transport properties, coefficients for the catalase reaction following a Michaelis-Menten type of kinetics were determined. Mean values of the Michaelis constant (k(M)) and the maximum rate of decompositon (v(max)) were k(M)=(85+/-55) mol m(-3) and v(max)=(49+/-40) nmol (s cell)(-1), respectively. The absolute values of P:(s) and sigma(s) of H(2)O(2) indicated that hydrogen peroxide, a molecule with chemical properties close to that of water, uses water channels (aquaporins) to cross the cell membrane rapidly. When water channels were inhibited with the blocker mercuric chloride (HgCl(2)), the permeabilities of both water and H(2)O(2) were substantially reduced. In fact, for the latter, it was not measurable. It is suggested that some of the water channels in CHARA: (and, perhaps, in other species) serve as 'peroxoporins' rather than as 'aquaporins'.

SummaryMechanisms that regulate water channels in the plant plasma membrane (PM) were investigated in Arabidopsis suspension cells. Cell hydraulic conductivity was measured with a cell pressure probe and was reduced 4-fold as compared to control values when calcium was added in the pipette and in bathing solution. To assess the signi®cance of these effects in vitro, PM vesicles were isolated by aqueous twophase partitioning and their water transport properties were characterized by stopped-¯ow spectrophotometry. Membrane vesicles isolated in standard conditions exhibited reduced water permeability (P f ) together with a lack of active water channels. In contrast, when prepared in the presence of chelators of divalent cations, PM vesicles showed a 2.3-fold higher P f and active water channels. Furthermore, equilibration of puri®ed PM vesicles with divalent cations reduced their P f and water channel activity down to the basal level of membranes isolated in standard conditions. Ca 2+ was the most ef®cient with a half-inhibition of P f at 50±100 mM free Ca 2+ . Water transport in puri®ed PM vesicles was also reversibly blocked by H + , with a half-inhibition of P f at pH 7.2±7.5. Thus, both Ca 2+ and H + contribute to a membrane-delimited switch from active to inactive water channels that may allow coupling of water transport to cell signalling and metabolism.

Hydroxyl radicals (*OH) as produced in the Fenton reaction (Fe 2 + + H 2 O 2 = Fe 3 + + OH -+ *OH) have been used to reversibly inhibit aquaporins in the plasma membrane of internodes of Chara corallina . Compared to conventional agents such as HgCl 2 , *OH proved to be more effective in blocking water channels and was less toxic to the cell. When internodes were treated for 30 min, cell hydraulic conductivity ( Lp ) decreased by 90% or even more. This effect was reversed within a few minutes after removing the radicals from the medium. In contrast to HgCl 2 , radical treatment reduced membrane permeability of small lipophilic organic solutes (ethanol, acetone, 1-propanol, and 2-propanol) by only 24 to 52%, indicating some continued limited movement of these solutes across aquaporins. The biggest effect of *OH treatment on solute permeability was found for isotopic water (HDO), which largely used water channels to cross the membrane. Inhibition of aquaporins reduced the diffusional water permeability ( P d ) by about 70%. For the organic test solutes, which mainly use the bilayer to cross the membrane, channel closure caused anomalous (negative) osmosis; that is, cells had negative reflection coefficients ( s s s s s ) and were transiently swelling in a hypertonic medium. From the ratio of bulk ( Lp or osmotic permeability coefficient, P f ) to diffusional ( P d ) permeability of water, the number ( N ) of water molecules that align in water channels was estimated to be N = P f / P d = 46 (on average). Radical treatment decreased N from 46 to 11, a value still larger than unity, which would be expected for a membrane lacking pores. The gating of aquaporins by *OH radicals is discussed in terms of a direct action of the radicals when passing the pores or by an indirect action via the bilayer. The rapid recovery of inhibited channels may indicate an easy access of cytoplasmic antioxidants to closed water channels. As hydrogen peroxide is a major signalling substance during different biotic and abiotic stresses, the reversible closure of water channels by *OH (as produced from H 2 O 2 in the apoplast in the presence of transition metals such as Fe 2 + or Cu + ) may be downstream of the H 2 O 2 signalling. This may provide appropriate adjustments in water relations (hydraulic conductivity), and a common response to different kinds of stresses.

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.

A review and re-examination of literature data shows that highly selective water channels (aquaporins) have marked effects on the overall transport properties of the-plasma membrane of plant cells. The application of the channel blocker HgCI 2 (50 //M) or of high external concentrations of permeating solutes reduced the water permeability (hydraulic conductivity, Lp) of Chara internodes down to 25% of the control. In treated cells, reflection coefficients (

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.

No abstract