Effects of sodium, potassium and calcium on salt-stressed barley. I. Growth analysis

Barley ( Hordeum vulgare L. cv. CM 72) was grown for a 28-day period and stressed with treatments of 125 mol m⁻³ NaCl or KC1 with low Ca²⁺ (0.4 mol m⁻³ Ca²⁺) or high Ca²⁺ (10 mol m⁻³ Ca²⁺). Plants were harvested periodically so that relative growth rate (RGR), net assimilation rate (NAR) and leaf area ratio (LAR) could be calculated using the functional approach to plant growth analysis. Relative growth rate declined with time for all treatments, including controls. Salinity inhibited RGR relative to control values by day 10. High Ca²⁺ improved the growth of salt-stressed plants in both NaCl-salinity and KCl-salinity. KC1 proved more toxic than NaCl, especially for KCI-salinity plants with low Ca²⁺, which died by day 28. Net assimilation rate, but not LAR, was highly correlated with RGR for all treatments. This indicates that the photosynthetic-assimilatory machinery was limiting RGR and not the leaf area of the plant.     

Supplemental manganese improves the relative growth, net assimilation and photosynthetic rates of salt-stressed barley

Previous results in our laboratory indicated that a reduced Mn concentration in the leaves of barley was highly correlated with the reduced relative growth and net assimilation rates of salt-stressed plants. If Mn deficiency limits the growth of salt-stressed barley, then increasing leaf Mn concentrations should increase growth. In the present study, the effect of supplemental Mn on the growth of salt-stressed barley ( Hordeum vulgare L. cv. CM 72) was tested to determine if a salinity-induced Mn deficiency was limiting growth. Plants were salinized with 125 mol m⁻³ NaCl and 9.6 mol m⁻³ CaCl₂. Supplemental Mn was applied in 2 ways: 1) by increasing the Mn concentration in the solution culture and 2) by spraying Mn solutions directly onto the leaves. Growth was markedly inhibited at this salinity level. Dry matter production was increased 100% in salt-stressed plants treated with supplemental Mn to about 32% of the level of nonsalinized controls. The optimum solution culture concentration was 2.0 mmol m⁻³, and the optimum concentration applied to the leaves was 5.0 mol m⁻³. Supplemental Mn did not affect the growth of control plants. Further experiments showed that supplemental Mn increased Mn concentrations and uptake to the shoot. Supplemental Mn increased the relative growth rate of salt-stressed plants and this increase was attributed to an increase in the net assimilation rate; there were no significant effects on the leaf area ratio. Supplemental Mn also increased the net photosynthetic rate of salt-stressed plants. The data support the hypothesis that salinity induced a Mn deficiency in the shoot, which partially reduced photosynthetic rates and growth.     

Effect of moderate salinity on patterns of potassium, sodium and chloride accumulation in cells near the root tip of barley: Role of differentiating metaxylem vessels

X-Ray microanalysis of fully hydrated, bulk-frozen samples was used to measure concentrations of potassium, sodium and chloride in various cell types along seminal roots of barley ( Hordeum vulgare L. cv. California Mariout) seedlings (1 to 150 mm from the tip). In the cytoplasm of all meristematic cells 1 mm from the root tip, the average concentrations of potassium and chloride were ca 200 and 15 m M , respectively. The potassium level was also high in the vacuoles of incipient xylem elements and did not drop to significantly lower values until 10 mm from the tip in protoxylem, 50 mm in early metaxylem and 150 mm in late metaxylem (LMX). Light microscopy observations (Nomarski optics) of hand-cut sections showed the presence of cytoplasmic strands and also the presence of intact cross walls in LMX up to a distance of 100 mm. Both quantitative analysis of ion contents and structural observations suggested that LMX elements act as a large transitional sink of accumulated ions and therefore may not function as a main pathway of transport until perforation of the end wall takes place 100–150 mm from the root tip. Treatment with 50 m M NaCl resulted in higher concentrations of sodium and chloride in LMX elements than in the surrounding cells, suggesting that living xylem elements, which develop a large central vacuole at an early stage of root differentiation, may assist in alleviating salinity stress in the meristematic region of barley root tips. Further, it is proposed that reabsorption of sodium and chloride from the LMX, especially before the disappearance of the cross walls, may provide a means of salinity tolerance.     

Na-Ca interactions in barley seedlings: relationship to ion transport and growth

Abstract Salt-stressed plants often show Ca deficiency symptoms. The effects of NaCl salinity (1 to 150 mol m^-3) and supplemental Ca (10 mol m^-3) on Na and Ca transport in barley (Hordeum vulgare L.) and their relationship to growth were investigated. The adjustment of Na and Ca transport was investigated by examining young seedlings exposed to short-term (immediate) and long-term (7 d) exposure to salinity. When the plants were exposed to long-term treatments of salinity, the rate of sodium accumulation in roots was approximately 10 to 15% of short-term treatments. No significant adjustment in the transport to the shoot was observed. Rates of tracer (²²Na) transport were compared to calculated rates based on relative growth rates and tissue element concentrations. Comparisons between measured tracer and calculated rates of transport indicate that ²²Na transport may underestimate transport to the shoot because of dilution of the tracer in the root cytoplasm. Calcium uptake showed only minor adjustment with time. Measured rates of tracer transport to the shoot correlated well with calculated values. The transport and tissue concentrations of Na were significantly affected by supplemental Ca. Calcium transport and tissue concentrations were markedly inhibited by salinity. Supplemental Ca increased Ca transport and accumulation at all NaCl treatments above that of control plants without supplemental Ca. Salinity inhibited plant growth at 150 mol m ^-3NaCl, but not at 75 mol m^-3. Supplemental Ca significantly improved root length but not fresh weight after 7d of salinity, although differences in fresh weight were detected after 9d. There were significant Na-Ca interactions with ion transport, ion accumulation, and growth. The effects of salinity on Na and Ca transport to the shoot do not appear to play a major role in shoot growth of barley.     

Effects of combined NaCl and CaCl2 salinity on photosynthetic parameters of barley grown in nutrient solution

The changes caused by NaCl− and CaCl₂-induced salinity on several leaf parameters have been measured in two cultivars of barley ( Hordeum vulgare L.) growing in a growth chamber in nutrient solution. Salinity was induced by adding to the nutrient solution equal weights of NaCl and CaCl₂, to obtain conductivities of 2, 6, 12, 19 and 26 dS m⁻¹. Salinity induced decreases in the leaf water potential and in the osmotic potential. Salinity did not induce significant changes in the relative photosynthetic pigment composition of barley leaves, the photosynthetic pigment stoichiometry for neoxanthin:violaxanthin cycle pigments:lutein:β-carotene:Chl b :Chl a being close to 3:6:14:12:25:100 (mol:mol). Salinity per se did not induce interconversions in the carotenoids within the violaxanthin cycle in most barley leaves. The PSII photochemistry of most barley leaves was unchanged by salinity. However, some apparently healthy leaves growing in high salinity exhibited sudden decreases in PSII photochemistry and increases in zeaxanthin (at the expense of violaxanthin), that preceded rapid leaf drying. Salinity induced significant changes in the slow part of the chlorophyll fluorescence induction curve from barley leaves.     

Inhibition of mugineic acid-ferric complex uptake in barley by copper, zinc and cobalt

The interfering effects of copper, zinc, and cobalt on the uptake of mugineic acid-ferric complex were studied in barley ( Hordeum vulgare , cv. Minorimugi) grown in nutrient solution. Short-term uptake experiments of 3 h were performed utilizing both ionic and mugineic acid-complex forms of each metal at two different concentrations. Copper was most effective in decreasing iron uptake when added in an ionic form at either concentration. The inhibition order at higher concentrations followed Cu(II) > Zn(II) ≥ Co(II), Co(III), which is consistent with the stability constants of these metal complexes with mugineic acid. The displacement of iron from its mugineic acid complex by these metals is suggested as a probable explanation for the decreased iron uptake. The inhibitory effect of metal complexes with mugineic acid on iron uptake was only found in cases with higher concentrations of Cu(II) and Zn(II) complexes. Deformation of the specific iron transport system in the plasma membrane due to their adsorption may be responsible for this effect.     

Histological analysis of somatic embryogenesis in Brazilian cultivars of barley, Hordeum vulgare vulgare, Poaceae

 Histological analysis was performed aimed at elucidating the origin and the developmental process of somatic embryos of two Brazilian cultivars of barley (Hordeum vulgare vulgare), 'MN-599' and 'A-05'. The observed site of somatic embryo origin (SSEO) could originate from a superficial callus cell, possibly indicating a unicellular origin, or from epidermal and subepidermal callus cells, representing a multicellular origin. A fold, the somatic embryo scutellum that subsequently develops into a cotyledonary leaf, indicates the somatic embryo differentiation. The somatic embryos also showed a growth increase of the primary root and, occasionally, a delay in root development. A possible alternative pathway for the origin of somatic embryos is suggested, in which a SSEO forms a clump of somatic embryos. Received: 4 June 1998 / Revision received: 28 August 1998 / Accepted: 7 December 1998     

Growth, contents of K+ and kinetics of K+(86Rb) uptake in barley cultured at different low supply rates of potassium

Plants of barley ( Hordeum vulgare L. cv. Salve) were grown with 6.5–35% relative increase of K⁺ supply per day (RKR) using a special computer-controlled culture unit. After a few days on the culture solution the plants adapted their relative growth rate (RGR) to the rate of nutrient supply. The roots of the plants remained in a low salt status irrespective of the rate of nutrient supply, whereas the concentration of K⁺ in shoots increased with RKR. Both V_max and K_m for K⁺(⁸⁶Rb) influx increased with RKR. It is concluded that with a continuous and stable K⁺ stress, the K⁺ uptake system is adjusted to provide an effective K⁺ uptake at each given RKR. Allosteric regulation of K⁺ influx does not occur and efflux of K⁺ is very small.     

Distribution and redistribution of sulphur taken up from nutrient solution during vegetative growth in barley

Barley plants were grown in a nutrient solution containing 25 μ M sulphate and the roots were pulsed with [³⁵S]sulphate for 48-h periods at 6 different times between the emergence of leaf 5 (L5) and the emergence of leaf 8 (L8). Growth was continued in unlabelled solution until the emergence of L10. Within the shoot system sulphur was directed principally into the leaf undergoing expansion. A large proportion of the ³⁵S-label delivered to young expanding leaves (> 40% of full expansion) did not occur at the time of the pulse, but subsequently during the ensuing chase indicating slow redistribution of sulphur from another site. During the later stages of leaf expansion (40–100%), most of the sulphur entered the leaf during the pulse, suggesting that sulphur was delivered more directly from the nutrient solution. Up to 75% of the sulphur delivered to L3–L6 at the time they approached or attained full expansion (70–100%) was re-exported. At least some of the sulphur exported from fully expanded leaves was redistributed to developing leaves.     

A comparison of barley isolated microspore and anther culture and the influence of cell culture density

Comparisons were made between the efficiency of barley plant regeneration from anther culture (AC) and isolated microspore culture (IMC) for the European winter cultivar `Igri' and the spring F<sub>1</sub> Australian breeder's hybrid Amagi Nijo×WI2585. In both cases, IMC produced a higher number of green regenerant plantlets per anther than AC. For `Igri' there was a 100- to 200-fold improvement and for Amagi Nijo×WI2585 there was a five- to ninefold improvement of IMC over AC. To improve the consistency and reliability of the IMC method, we investigated several parameters, including maltose concentration, subculture protocol, microspore plating density and colony plating density. Subculturing during the liquid culture phase produced no significant improvement in the number of microspores developing into colonies. The optimal concentration of maltose in the liquid induction medium was found to be 90 g l^–1. Both microspore plating density and colony plating density were found to influence plant regeneration. Microspores produced the highest numbers of colonies when plated at densities greater than 5×10⁴ ml^–1, and colonies produced optimal numbers of green plantlets when plated at 12.5–25 colonies/cm². Received: 23 March 1997 / Revision received: 29 May 1997 / Accepted: 25 June 1997     

Nitrate assimilatory properties of barley grown under long-term N limitation: Effects of local nitrate supply in split-root cultures

The effect of nitrate availability on characteristics of the nitrate assimilatory system was investigated in N-limited barley (Hordeum valgare L. cv. Golf), grown with the seminal root system split into initially equal-sized halves. The cultures were continuously supplied with nitrate-N at a relative addition rate (RA) of 0.09 day^?1, which resulted in relative growth rates (RG) that were ca 85% of those observed under surplus nitrate nutrition. The total N addition was divided between the subroots in ratios of 100:0, 80:20, 70:30, 60:40, and 50:50. For comparison, standard cultures were grown at RAs ranging from 0.03 to 0.18 day^?1. Initially, biomass and N partitioning to the subroots responded strongly and proportionally to the nitrate distribution ratio. After 12-14 days no further effect was observed. The V_max for net nitrate uptake and in vitro nitrate reductase (NR) activity were measured in acclimated plants, i.e., after > 14 days under a certain nitrate regime. In subroots fed from 20 to 100% of the total N addition, V_max for net nitrate uptake increased slightly, whereas NR activity was unaffected. Uptake and NR activities were insignificant in the 0%-subroot. Uneven nitrate supply to individual subroots had negligible effect on the whole-plant ability for nitrate uptake, and the relative V_max (unit N taken up per unit N in whole plant tissue and time) remained about 7-fold in excess of the demand set by growth. Balancing nitrate concentrations (the resulting external nitrate concentrations at a certain RA) generally ranged between 2 and 10 μM at growth-limiting RA, both when predicted from uptake kinetics and when actually measured. When comparing split root and standard cultures when acclimated, it appears that uptake and NR activities in roots respond more strongly to over-all nitrate availability than to nitrate availability to individual subroots.     

Effect of calcium on sulfate uptake by barley roots

Abstract Sulfate uptake by excised roots of barley (Hordeum vulgare L.) was maximal in the presence of about 3x10^-3M CaCl₂. Kinetic studies contraindicate a stoichiometric binding of calcium to the carrier for sulfate, in contrast to findings of Cuppoletti and Segel (Biochemistry 14: 471–4718, 1975) for the filamentous fungus Penicillium notatum. In barley, calcium affects the K_m but not the V_max for sulfate uptake, presumably by altering the conformation and, thereby, the affinity of the carrier. Calcium also affects the transition site for sulfate uptake.     

Changes in the protein composition of cytoplasmic ribsomes during the greening of etiolated barley leaves

We examined changes in the protein composition of cytoplasmic ribosomes in etiolated barley leaves following illumination. Cytoplasmic ribosomes were isolated from greening barley leaves by sucrose density gradient centrifugation, and were analyzed by radical-free highly reducing polyacrylamide gel electrophoresis (RFHR-PAGE). Eighty-nine proteins were resolved from the ribosomal fraction; among them, 8 proteins changed their copy numbers depending on the stage of greening. We designated these as phase dependent ribosomal proteins (PD1–PD8). Two of the proteins (PD1 and 5) present in the ribosomes of etiolated leaves showed a decrease in level during greening. In contrast, the levels of 6 ribosomal proteins (PD2, 3, 4, 6, 7 and 8) increased as greening proceeded. N-terminal amino acid sequence of PD8 showed high homology to rat ribosomal protein L34. The ribosomal proteins that appeared after illumination were not found in any fraction of the etiolated leaves, suggesting that they were synthesized after the onset of illumination. Copy numbers of other ribosomal proteins did not change during greening.     

Translocation of N to and from barley roots: its dependence on local nitrate supply in split-root culture

The relationship between availability of external nitrate and N translocation between root and shoot was studied in N-limited barley ( Hordeum vulgare L. cv. Golf). Nitrate-N was added at a relative rate (i.e. N added per unit time and unit N in plant biomass) of (1.09 da-¹, and distributed between the subroots at ratios of 50:50 or 80:20. The plants were grown for 13 days under these conditions of nitrate nutrition, and for another three days with the nitrate distribution reversed from 80:20 to 20:80. The nitrale-N doses thus experienced by individual subroots ranged from 2 to 11 mg N g-¹ root dry weight day-¹ . ¹⁵N-Nitrate labellings were performed after 2 to 3 and 12 to 13 days of nitrate nutrition. and 2 to 3 days after reversal of nitrate additions. For all treatments, between 60 and 82% of the absorbed label initially left the root, and between 25 and 55% of the label recovered in roots had been supplied (cycled) via the shoot. Labelling of xylem N at the end of the 24-h labelling period ranged from to 36 to 46% indicating that a substantial fraction of the N in the xylem had been absorbed by the plant prior to labelling. It is concluded that cycling of N to roots, and cycling of N in the plant as a whole, is substantial also during N-limited growth. N allocation to roots increased with external nitrate dose. An increased utilization of non-translocated N as well as an increased translocation of N from the shoot contributed to this effect. Thus, the results indicate that increased external availability of N also increased the sink strength of the root for cycling N.     

NADH-dependent nitrate reductase from two-row barley roots: Purification, characteristics and comparison with leaf enzyme

NADH-nitrate reductase (EC 1.6.6.1) was purified 800-fold from roots of two-row barley ( Hordeum vulgare L. cv. Daisen-gold) by a combination of Blue Sepharose and zinc-chelate affinity chromatographies followed by gel filtration on TSK-gel (G3000SW). The specific activity of the purified enzyme was 6.2 μmol nitrite produced (mg protein)⁻¹ min⁻¹ at 30°C.
Besides the reduction of nitrate by NADH, the root enzyme, like leaf nitrate reductase, also catalyzed the partial activities NADH-cytochrome c reductase, NADH-ferricyanide reductase, reduced methyl viologen nitrate reductase and FMNH₂-nitrate reductase. Its molecular weight was estimated to be about 200 kDa, which is somewhat smaller than that for the leaf enzyme. A comparison of root and leaf nitrate reductases shows physiologically similar or identical properties with respect to pH optimum, requirements of electron donor, acceptor, and FAD, apparent K_m for nitrate, NADH and FAD, pH tolerance, thermal stability and response to inorganic orthophosphate. Phosphate activated root nitrate reductase at high concentration of nitrate, but was inhibitory at low concentrations, resulting in increases in apparent K_m for nitrate as well as V_max whereas it did not alter the K_m for NADH.     

Regulation of phosphate influx in barley roots: Effects of phosphate deprivation and reduction of influx with provision of orthophosphate

Barley plants were grown in nutrient solutions, which were maintained at either 0 (-P) or 15 μ M orthophosphate (+P). After 11 days phosphate influx into the intact roots of the -P plants began to increase by comparison with +P plants. During this period differences became apparent between the treatments in absolute growth rates, as well as in the root:shoot ratios. Phosphate influx in the -P plants continued to increase as a function of time, to a maximum value of 2.4 μmol (g fresh wt)^-1h^-1 at 16 days after germination. This rate was 6 times higher than influx values for +P plants of the same age. During the period of enhanced uptake phosphate was strongly correlated (r²= 0.77) with root organic phosphate concentration. – The enhancement of inorganic phosphate influx into intact roots of -P plants was rapidly reduced by the provision of 15 μ M orthophosphate. Typically, within 4 h of exposure to this concentration of phosphate, influx values fell from 1.80 ± 0.20 to 0.75 ± 0.03 μmol (g fresh wt)^-1 h^-1, while inorganic phosphate concentrations of the roots increased from 0.12 to 1.15 μmol (g fresh wt)^-1 during the same period. Hill plots of the influx data obtained during this period, treating root inorganic phosphate as an inhibitor of influx, gave Hill coefficients close to 2. The rapidity of the reduction of influx associated with increased root inorganic phosphate together with the Hill plot data provide evidence for an allosteric inhibition of influx by internal inorganic phosphate.     

Effects of UV-B on flavonoids, ferulic acid, growth and photosynthesis in barley primary leaves

Barley (Hordeum vulgare L.) was grown with UV-B (280–320 nm) at levels simulating 25 nr 5% ozone depletion on the date of the summer solstice al 40°N latitude, with UV-A (320–400 nm), or with no supplemental irradiation. In plant growth chambers providing 300 μmol m^?2 s^?1 photosynthetically active radiation (PAR). UV-B-grown leaves elongated more slowly than controls but reached the same final length 1 day later. Leal specific fresh weight (mass leaf area^?1) was significantly increased by UV-B after the 7th day of growth. IV-B did not significantly affect leaf area, fresh weight, dry weight, total chlorophylls, total carotenoids or photosynthetic quantum efficiency. CO₂ assimilation was decreased by UV-B only at internal CO₂ levels above 250 μl l^?1. By the 8th day of growth, UV-B increased flavonoid (saponarin and lutonarin) accumulation in both the lower epidermis and the mesophyll: about 40% of the saponarin and 20% of the lutonarin were in the lower epidermis under all experimental conditions. Glasshouse conditions proved too variable for reproducible determination of growth and photosynthesis but were reliable for determining developmental changes in flavonoid (saponarin and lutonarin) accumulation and provided up to 800 μmol m^?2 s^?1 PAR. In the glasshouse UV-B-grown leaves had more flavonoids than controls al all stages from 5 to 30 days after planting: ca 509 more saponarin and 100% more lutonarin. Levels of soluble (vacuolar) ferulic acid esters were similar under all conditions on day 5. and on day 20 or later, but were significantly higher in UV-B-grown plants on days 10 and 15. UV-B decreased insoluble (cell-wall-bound) ferulic acid esters on a whole leaf basis but significantly increased this fraction in the lower epidermis. UV-A had no significant effects on growth, photosynthesis or ferulic acid, but it slightly increased flavonoid accumulation. The results are discussed in terms of secondary phenolics as a tissue-specific, developmentally regulated adaptive response to UV-B.     

Quantitative changes in potassium,sodium, and calcium in the submaxillary salivary gland and blood serum of rats exposed to 2880-MHz microwave radiation

Na⁺, K⁺, and Ca²⁺ concentrations in the blood serum and submaxillary salivary gland (SSG) were investigated in adult, male rats exposed to 2880-MHz microwaves modulated with 1.5-μs pulses at a pulse repetition rate of 1000 Hz or in a hyperthermal environment. Rats were exposed, one at a time, for 30 min to microwaves producing a specific absorption rate (SAR) of: 4.2, 6.3,6.8,8.4, 10.8, or 12.6 W/kg, or were sham exposed under similar environmental conditions. In a second series, one group of rats was exposed singly for 15, 30, or 60 min to microwaves producing an SAR of 9.5 W/kg and other rats were exposed for similar periods at 40 °C; and 10 rats were sham exposed. Flame photometric analysis indicated that the thresholds of microwave radiation required to induce a change in Na⁺, K⁺, and Ca²⁺ concentrations in the salivary glands are 6.8, 6.8, and 6.3 W/kg, respectively. The directions of Na⁺, K⁺, and Ca²⁺ ion shifts in exposed rats' salivary glands are similar, whether affected by microwaves or hyperthermia. Greater changes in Na⁺ and K⁺ concentrations in SSG of rats exposed to microwaves for 15 and 30 min were found than in those exposed at 40 °C. On the other hand, exposure to hyperthermia at 40 °C or to microwaves for 1 h caused Na⁺ concentration to be increased by 68.7 and 59.5% and K⁺ concentration to be decreased by 29.6 and 21.7%, respectively.     

Effects of Russian wheat aphid infestation on barley plant response to drought stress

The influence of Russian wheat aphid ( Diuraphis noxia Mordvilko) infestation on the response of barley ( Hordeum vulgare L. ev Hazen) plants to drought stress was investigated. Fourteen-day-old plants were infested with eight apterous adult aphids, which were removed 7 days later with systemic insecticide. Leaves previously infested with aphids had lower relative water content, reduced stomatal conductance, more negative water potential, lower levels of chlorophyll and higher levels of amino-N, proline and glycinebetaine than corresponding leaves from uninfested plants. When water was withheld for a period of 7 days after aphids were removed, the relative water content of previously infested plants dropped steadily from 0.89 to 0.60, while the relative water content of uninfested plants remained at about 0.94 for the first 4 days of the drought stress period followed by a steady drop to about 0.77 by the end of the drought stress period. Leaf water potentials dropped steadily during the drought stress period in both previously infested (-1.14 to -1.91 MPa) and unin-fested (-0.54 to -1.52 MPa) plants. Analysis of glycinebetaine and proline levels at the end of the drought stress period indicated that leaves of previously infested plants accumulated lower levels of these solutes than leaves from uninfested plants. Upon alleviation of drought stress, plants previously infested with aphids showed little increase in dry weight while younger leaves and tillers from uninfested plants showed large increases. It is concluded that Russian wheat aphids cause drought-stress symptoms in leaves of infested plants even in the presence of ample root moisture. The observations of low levels of glycinebetaine and proline present in leaves after water was withheld from roots and lack of leaf growth upon alleviation of drought stress in previously-infested plants, suggest that aphid infestation limits the capacity of barley plants to adjust successfully to drought stress.