R. Gareth Wyn Jones

Assessment of glycinebetaine and proline compartmentation by analysis of isolated beet vacuoles

Vacuoles isolated from storage root tissue of red beet (Beta vulgaris L.) do not leak significant quantities of betanin, sucrose, Na+ or K+ during isolation. This indicates that analysis of vacuoles in vitro gives meanigful information about the compartmentation of solutes in vivo. Preparations of vacouoles were used to determine the distribution of glycinebetaine and proline between vacuole and cytoplasm in beet cells. Both compounds were detected in preparations of isolated beet vacuoles. In the case of glycinebetaine it was shown that this solute was associated with the vacuoles, not with the small number of other organelles which contaminated the preparations. The vacuolar pool accounted for 26 to 84% of the total tissue glycinebetaine and 17 to 57% of the proline. Concentrations of these compounds in vacuole and cytoplasm were calculated and were always higher in the cytoplasm than in the vacuole. The concentration gradient across the tonoplast varied considerably. The significance of these results is discussed in relation to the hypothesis that glycinebetaine and proline function as benign cytoplasmic osmotica.

The effect of abscisic acid on cell turgor pressures, solute content and growth of wheat roots

Abscisic acid (ABA) was shown to influence turgor pressure and growth in wheat (Triticum aestivum L.) roots. At a concentrations of 25 mmol·m-3, ABA increased the turgor pressure of cells located within 1 cm of the tip by up to 450 kPa. At 4 to 5 cm from the root tip this concentration of ABA reduced the turgor pressure of peripheral cells (epidermis and the first few cortical cell layers) to zero or close to zero while that of the inner cells was increased. Increases in sap osmolality were dependent on the concentration of ABA and the effect saturated at 5 mmol·m-3 ABA. The increase in osmolality took about 4 h and was partly the result of reducing-sugar accumulation. Levels of inorganic cations were not affected by ABA. Root growth was inhibited at ABA concentrations that caused a turgor-pressure increase. The results show that while ABA can affect root cell turgor pressures, this effect does not result in increased root growth.

Glycinebetaine biosynthesis and its control in detached secondary leaves of spinach

In secondary leaves from spinach plants pretreated in vermiculite for 24 h with 300 mM NaCl, glycinebetaine accumulated at a rate of circa 0.16 μmol 100 μg-1 Chl d-1 (2 μmol g-1 FW d-1), about three times the rate of control plants. The soluble carbohydrate and free amino acid contents did not increase significantly following salinisation until after 4 d when the relative growth rate also decreased. Leaf proline levels remained very low throughout the experimental period. K+ on a tissue water basis remained constant at 200 mM while Cl- and Na+ levels increased linearly to reach 175 and 100 mM respectively after 5 d of saline treatment. The osmotic pressure of leaf tissue also increased from 300 to 500 mosmol kg-1. These experimental conditions were considered suitable to study glycinebetaine biosynthesis and its induction by salinity in the absence of marked growth inhibition or metabolic disturbance. Radioactive labelled [14C]serine, ethanolamine and choline (all 1 μmol, 13.3 MBq in 10 μl) were fed to detached secondary leaves via the petiole 24 h after the exposure of plants to salt. The rate of isotope incorporation into water soluble products, lipids and residue was measured over a further 24 h. The major metabolic fate of exogenous [14C]choline and [14C]ethanolamine was incorporation into glycinebetaine while less 14C-label was found in phosphatidyl choline and phosphatidyl ethanolamine. Incorporation rates were identical in control and salinised leaves and were adequate to account for observed values of glycinebetaine accumulation previously reported in spinach. In contrast the labelling of glycinebetaine from [14C]serine was twice as great in salinated plants as in the controls. These results, together with short term labelling experiment with [14C]ethanolamine using leaf slices, were consistent with the formation of glycinebetaine via serine, ethanolamine and its methylated derivatives to choline with some control being exerted at the serine level. However a flux through the phosphorylated intermediates is not excluded.From a consideration of these results and the published data on barley subjected to water stress (Hanson and Scott, 1980 Plant Physiol. 66, 342–348) there appear to be significant differences in the biosynthetic pathways in spinach and barley.

Water-relation parameters of epidermal and cortical cells in the primary root ofTriticum aestivum L.

Water-relation parameters of root hair cells, hairless epidermal cells, and cortical cells in the primary root of wheat have been measured using the pressure-probe technique. Under well-watered conditions the mean cell turgor of cortical cells was 6.8±1.9 (30) bar (mean±SD; the number of observations in brackets). In hairless epidermal and root hair cells the mean cell turgor was 5.5±1.9 (22) and 4.4±1.5 (15) bar, respectively. Despite the large variability, turgor pressure was significantly lower (confidence interval=0.95) in epidermal cells relative to cortical cells. This may be a consequence of the ultrafiltration of ions by the external cell wall and-or plasmalemma of epidermal cells. The volumetric elastic modulus of the cells ranged from 10 to 150 bar. This parameter was dependent on cell volume, but within experimental accuracy, was independent of cell type. No pressure dependence of the volumetric elastic modulus was observed in these cells. The half-times for water exchange ranged from 1.8 to 48.8 s. The mean value increased in the order root hair < hairless epidermal < cortical cells and was directly related to volume to surface area ratio. Thus the hydraulic conductivities of the three cell types were similar and averaged 1.2±0.9·10-6 (170) cm s-1 bar-1. No polarity was observed between inwardly and outwardly directed water flow. The similarity of the hydraulic conductivities of root hairs to those of other cells indicates that the membranes of root hairs are not particularly specialized for water transport. The overall hydraulic conductivity for radial water flow across the root was estimated from the pressure-probe data using a simple model and was compared with that measured directly on whole roots using an osmotic backflow technique. It was tentatively concluded that upon sudden osmotic perturbation, the major pathway for water transfer across the root may be through the symplasm and involve net flow from vacuole to vacuole.

The integration of whole-root and cellular hydraulic conductivities in cereal roots

The hydraulic conductivities of excised whole root systems of wheat (Triticum aestivum L. cv. Atou) and of single excised roots of wheat and maize (Zea mays L. cv. Passat) were measured using an osmotically induced back-flow technique. Ninety minutes after excision the values for single excised roots ranged from 1.6·10-8 to 5.5·10-8 m·s-1·MPa-1 in wheat and from 0.9·10-8 to 4.8·10-8 m·s-1·MPa-1 in maize. The main source of variation was a decrease in the value as root length increased. The hydraulic conductivities of whole root systems, but not of single excised roots, were smaller 15 h after excision. This was not caused by occlusion of the xylem at the cut end of the coleoptile. The hydraulic conductivities of epidermal, cortical and endodermal cells were measured using a pressure probe. Epidermal and cortical cells of both wheat and maize roots gave mean values of 1.2·10-7 m·s-1·MPa-1 but in endodermal cells (measured only in wheat) the mean value was 0.5·10-7 m·s-1·MPa-1. The cellular hydraulic conductivities were used to calculate the root hydraulic conductivities expected if water flow across the root was via transcellular (vacuole-to-vacuole), apoplasmic or symplasmic pathways. The results indicate that, in freshly excised roots, the bulk of water flow is unlikely to be via the transcellular pathway. This is in contrast to our previous conclusion (H. Jones, A.D. Tomos, R.A. Leigh and R.G. Wyn Jones 1983, Planta 158, 230–236) which was based on results obtained with whole root systems of wheat measured 14–15 h after excision and which probably gave artefactually low values for root hydraulic conductivity. It is now concluded that, near the root tip, water flow could be through a symplasmic pathway in which the only substantial resistances to water flow are provided by the outer epidermal and the inner endodermal plasma membranes. Further from the tip, the measured hydraulic conductivities of the roots are consistent with flow either through the symplasmic or apoplasmic pathways.

Utilization of Triticeae for improving salt tolerance in wheat

By the use of a wide range of genetic materials in experiments on the comparative physiology of salt tolerance and ion uptake in annual and perennial members of the tribe Triticeae, we have identified three physiological mechanisms which may contribute to salt tolerance. These are (a) enhanced K/Na discrimination in transport of ions to the shoots (from Aegilops squarrosa), (b) improved total salt exclusion at high salinities (from Thinopyrum bessarabicum) and (c) tolerance of high leaf salt concentrations (in barley). The identification of these very different mechanisms supports the idea that improved salt tolerance in wheat may be achieved by pyramiding, i.e. isolation of individual characters, understanding (modelling) of their function and interrelations, design of ideal salt-tolerant idiotypes based on these models and ultimately integration of individual characters into crops with increased salt tolerance.

Calcium-accumulating cells in the meristematic region of grapevine root apices

Apical roots of grapevines were examined by cryo-SEM (scanning electron microscopy) and the intracellular distribution of Ca was demonstrated by X-ray microanalysis in different regions of the primary root. We show that large amounts of Ca are accumulated as raphide crystals in the vacuoles of specialised cortical cells (idioblast cells) of the root apex. These crystal idioblast cells appeared to form a discontinuous cone of cells in the outer region of the root meristem. The raphide crystals within these cells were less apparent in older regions of the root, 10-12 mm basipetal to the root tip. We suggest that the raphide crystals could initially act as another Ca sink involved in the regulation of Ca levels in root apices. In older regions of the root these cells are spaced at intervals around the periphery of the cortex and the subsequent disappearance of the raphides may be indicative of remobilisation, perhaps in the zone of elongation where cell wall synthesis occurs and Ca demand is high. Calcium-accumulating cells were also observed in the older regions of the root, forming endodermal protrusions extending into the cortex. These cells may play a part in regulating Ca delivery to the xylem stream by sequestration of Ca from the radial flow of water at the endodermis. The observed distribution of Ca in root apices was different from the other major cations (e.g. K) and anions (e.g. Cl) because high concentrations were localised to specific cells. We interpret the results in the context of a model of the dynamics of grapevine root growth and cell differentiation, and the temporal balance of solute supply from the protophloem and the external medium.