Production and organ distribution of succinate in rice seedlings during anoxia

Anaerobic production of succinate, a common feature in animals able to sustain anoxia, has seldom been reported in plants. By the use of ¹H-nuclear magnetic resonance spectroscopy we show here that succinate is produced by rice seedlings (Oryza sativa L. cv. Arborio) subjected to anoxic conditions. Starting from levels below I μmol (g fresh weight)⁻¹ in air, after 48 h of anoxia the levels of alanine, succinate and lactate had increased to 23.8, 5.2 and 1.0 μmol (g fresh weight) ⁻¹, respectively, in shoot tissues. Succinate was accumulated in shoots, notably in the coleoptiles, but not in roots of the rice seedlings, suggesting its involvement in rice coleoptile elongation under anoxia. Other possible functions of succinate production in rice seedling, an organism highly tolerant to anoxia, are discussed.     

Effect of oxygen concentration on intracellular pH, glucose-6-phosphate and NTP content in rice (Oryza sativa) and wheat (Triticum aestivum) root tips: in vivo 31P-NMR study

Hypoxic pretreatment is known to induce anoxia tolerance in plant species sensitive to oxygen deprivation. However, we still do not have detailed information on changes in cytoplasmic and vacuolar pH (pH_cyt and pH_vac) in plants under low-oxygen availability (hypoxia) and under anoxia. To investigate this, we have studied the influence of hypoxia and anoxia on pH_cyt and pH_vac, glucose-6-phosphate (Glc-6-P) and nucleotide triphosphate (NTP) contents in rice ( Oryza sativa L.) root tips in comparison with those of wheat ( Triticum aestivum L.) with in vivo ³¹P-nuclear magnetic resonance. Both cereals responded to hypoxia similarly, by rapid cytoplasmic acidification (from pH 7.6–7.7 to 7.1), which was followed by slow partial recovery (0.3 units after 6 h). Anoxia led to a dramatic pH_cyt drop in tissues of both species (from pH 7.6–7.7 to less than 7.0) and partial recovery took place in rice only. In wheat, the acidification continued to pH 6.8 after 6 h of exposure. In both plants, NTP content followed the dynamics of pH_cyt. There was a strong correlation between NTP content and cytoplasmic H⁺ activity ([H⁺]_cyt= 10^−pHcyt) for both hypoxic and anoxic conditions. Glc-6-P content increased in rice under anoxia and hypoxia. In wheat, Glc-6-P was not detectable under anoxia but increased under hypoxia. In this study, rice root tips were shown to behave as anoxia tolerant tissues. Our results suggest that the initial cytoplasmic acidification and subsequent pH_cyt are differently regulated in anoxia tolerant and intolerant plants and depend on the external oxygen concentration.     

Nutrient translocation pattern and accumulation of free amino acids in rice coleoptile elongation under anoxia

Comparing nutrient translocation to the rice ( Oryza sativa L. var. Arborio ) shoot during anoxia with the aerobic situation, it was found that anoxia reduced the translocation of K⁺, phosphorus, Mg²⁺ and Ca²⁺ with progressive intensity; Ca²⁺ translocation was practically zero in the absence of oxygen. The translocation of K⁺ and phosphorus under anoxia was still considerable and contributed to the maintenance of a high osmotic potential while the blocking of Ca²⁺ translocation caused a decrease in its concentration in the anoxic coleoptile, possibly favouring high cell wall plasticity in that organ. As anoxia proceeded, amino acids, no longer employed in protein synthesis, accumulated in the coleoptile, reaching spectacular levels [51 mmol kg of tissue-water)⁻¹] and, after 48 h of anoxia, their contribution to the osmotic potential was 80% of that of K⁺, as against less than 20% in all aerobic treatments. Anoxia caused a reduction in soluble hexose concentrations which, however, were more than compensated osmotically by the accumulation of amino acids.     

A comparison of NH4+ and NO3– net fluxes along roots of rice and maize

Net fluxes of NH₄⁺ and NO₃^– along adventitious roots of rice ( Oryza sativa L.) and the primary seminal root of maize ( Zea mays L.) were investigated under nonperturbing conditions using ion-selective microelectrodes. The roots of rice contained a layer of sclerenchymatous fibres on the external side of the cortex, whereas this structure was absent in maize. Net uptake of NH₄⁺ was faster than that of NO₃^– at 1 mm behind the apex of both rice and maize roots when these ions were supplied together, each at 0·1 mol m^–3. In rice, NH₄⁺ net uptake declined in the more basal regions, whereas NO₃^– net uptake increased to a maximum at 21 mm behind the apex and then it also declined. Similar patterns of net uptake were observed when NH₄⁺ or NO₃^– was the sole nitrogen source, although the rates of NO₃^– net uptake were faster in the absence of NH₄⁺. In contrast to rice, rates of NH₄⁺ and NO₃^– net uptake in the more basal regions of maize roots were similar to those near the root apex. Hence, the layer of sclerenchymatous fibres may have limited ion absorption in the older regions of rice roots.     

Wheat genotypes show contrasting abilities to recover from anoxia in spite of similar anoxic carbohydrate metabolism

Physiological and metabolic responses to anoxia and reaeration were compared for 4–7-day-old seedlings of 11 genotypes of wheat (Triticum aestivum) with reputed differences in waterlogging tolerance. Genotypes differed in seminal root elongation, and recovery of root tissue K⁺ concentration, during reaeration following 72 h anoxia. Post-anoxic recovery ranged from complete (100% retention of seminal root elongation potential) to almost nil (death of all seminal root apices and inability to recover K⁺ concentration). The anoxia tolerance ranking of the genotypes based on these parameters corresponded with that of their reputed waterlogging tolerance, but with some exceptions. However, the differences in anoxia tolerance of the seedlings could not be explained by differences in capacity for ethanol production. A decreased ability to utilise seed starch reserves under anoxia, due to inadequate levels of -amylase activity at the time anoxia was imposed, was apparent in all genotypes.     

Calcium ion dependency of ethylene production in segments of primary roots of Zea mays

We investigated the effect of Ca²⁺ on ethylene production in 2-cm long apical segments from primary roots of corn ( Zea mays L., B73 × Missouri 17) seedlings. The seedlings were raised under different conditions of Ca²⁺ availability. Low-Ca and high-Ca seedlings were raised by soaking the grains and watering the seedlings with distilled water or 10 m M CaCl₂, respectively. Segments from high-Ca roots produced more than twice as much ethylene as segments from low-Ca roots. Indoleacetic acid (IAA; 1 μ M ) enhanced ethylene production in segments from both low-Ca and high-Ca roots but auxin-induced promotion of ethylene production was consistently higher in segments from high-Ca roots. Addition of I-aminocyclopropane-I-carboxylic acid (ACC) to root segments from low-Ca seedlings doubled total ethylene production and the rate of production remained fairly constant during a 24 h period of monitoring. In segments from high-Ca seedlings ACC also increased total ethylene production but most of the ethylene was produced within the first 6 h. The data suggest that Ca²⁺ enhances the conversion of ACC to ethylene. The terminal 2 mm of the root tip were found to be especially important to ethylene biosynthesis by apical segments and, experiments using ⁴⁵Ca²⁺ as tracer indicated that the apical 2 mm of the root is the region of strongest Ca²⁺ accumulation. Other cations such as Mn²⁺, Mg²⁺, and K⁺ could largely substitute for Ca²⁺. The significance of these findings is discussed with respect to recent evidence for gravity-induced Ca²⁺ redistribution and its relationship to the establishment of asymmetric growth during gravitropic curvature.     

Cadmium and copper change root growth and morphology of Pinus pinea and Pinus pinaster seedlings

Heavy metal loads in forest soils have been increasing over time due to atmospheric inputs. Accumulation in the upper soil layers could affect establishment of seedlings and forest regeneration. Mediterranean species show a high initial root development, allowing seedlings to reach the moisture of deeper soil layers. In the present work seedlings of stone pine ( Pinus pinea L.) and maritime pine ( Pinus pinaster Ait.), were grown in culture solution supplied with 0.0, 0.1, 1 or 5 μ M CdSO₄ or with 1 μ M CdSO₄ and 1 μ M CuSO₄ combined. In both species tap-root elongation was drastically reduced in the 5 μ M Cd²⁺ and in the (Cd²⁺+ Cu²⁺) treatments. A supply of 0.1 or 1 μ M Cd²⁺, however, enhanced root elongation in Pinus pinea without significantly influencing root elongation in Pinus pinaster . In both species the root density (weight per unit length) and the width of the cortex increased in response to Cd²⁺ exposure. In Pinus pinaster the mitotic index decreased at the higher Cd²⁺ concentrations and when Cd²⁺ and Cu²⁺ were combined. The data suggest that cell elongation is more sensitive to Cd²⁺ than cell division. The number and length of the lateral roots were also affected by Cd²⁺ treatment to a higher degree in Pinus pinaster than in Pinus pinea, reflecting the different Cd- tolerance of the two species.     

Promotive effect of capillarol and related compounds on root growth

The promotion of root growth by capillarol [methyl 3-(3-methylbut-2-enoyl)-4-hydroxycinnamate] and related phenolic compounds were studied in relation to structure-activity relationships. Concentrations above 5 × 10⁻⁵ M capillarol stimulated the root growth of rice ( Oryza sativa L. cv. Tanginbozu and cv. Nihonbare) seedlings to about 180% of the control value at 5 × 10⁻⁴ M . Capillarol had no promotive and hardly any inhibitory effect on the growth of the second leaf sheath. Capillarol promoted the root growth also in seedlings of lettuce ( Lactuca sativa L. cv. Grand Rapids) to ca 150% of the control value at 5 × 10⁻⁴ M . The free acid form of capillarol (capillaric acid) was about as effective as capillarol. Para -hydroxy- but not m -methoxy- substituted cinnamic acid, phenylpyruvic acid, phenylacetic acid and amino-hydrocinnamic acid could stimulate root growth, but p -hydroxybenzoic acid was inactive. It is concluded that the important structural requirements for high root growth-promoting activity of phenolic compounds are the hydroxyl group substitution at the C-4 position of the benzene ring, and the propanoic or propenoic acid side chain at the C-1 position. A possible mode of the action of capillarol on root growth-promoting activity is also discussed.     

Different effects of supplied ammonium on glutamine synthetase activity in mustard (Sinapis alba) and pine (Pinus sylvestris) seedlings

The activity of glutamine synthetase (GS) in mustard ( Sinapis alba L.) and Scots pine ( Pinus sylvestris L.) seedlings was used as an index to evaluate the capacity to cope with excessive ammonium supply. In these 2 species GS activity was differently affected by the application of nitrogen compounds (NH₄⁺ or NO₃⁻). Mustard seedlings older than 5 days showed a considerable increase in GS activity after NH₄⁺ or NO₃⁻ application. This response was independent of the energy flux, but GS activity in general was positively affected by light. Endogenous NH₄⁺ did not accumulate greatly after nitrogen supply. In contrast, seedlings of Scots pine accumulated NH₄⁺ in cotyledons and roots and showed no stimulation of GS activity after the application of ammonium. In addition, root growth was drastically reduced. Thus, the pine seedlings seem to have insufficient capacity to assimilate exogenously supplied ammonium. NO₃⁻, however, did not lead to any harmful effects.     

Abscisic acid accumulation and cadmium tolerance in rice seedlings

Rice ( Oryza sativa L.) seeds were soaked for 18 h in distilled water in the absence (–PBZ) or presence (+PBZ, a triazole) of 100 mg l⁻¹ paclobutrazol and then air dried. These air-dried seeds were germinated in the dark and then cultivated in a Phytotron. Twelve-day-old –PBZ and +PBZ seedlings were treated or not with CdCl₂. Cd toxicity was judged by the decrease in biomass production, decrease in chlorophyll and protein content, increase in NH₄⁺ content and induction of oxidative stress. The results indicated that PBZ applied to seeds was able to protect rice seedlings from Cd toxicity. On treatment with CdCl₂, the abscisic acid (ABA) content increased in +PBZ leaves, but not in –PBZ leaves. The decrease in the transpiration rate of –PBZ seedlings by CdCl₂ was less than that of +PBZ seedlings. Exogenous application of the ABA biosynthesis inhibitor, fluridone (Flu), reduced ABA accumulation, increased the transpiration rate and Cd content, and decreased the Cd tolerance of +PBZ seedlings. The effects of Flu on the Cd toxicity, transpiration rate and Cd content were reversed by the application of ABA. It seems that the PBZ-induced Cd tolerance of rice seedlings is mediated through an accumulation of ABA.     

Exposure of roots of cucumber (Cucumis sativus) to low temperature severely reduces root pressure, hydraulic conductivity and active transport of nutrients

When root temperature dropped below 25°C, there was a sharp drop in the root pressure ( P _r) and hydraulic conductivity of excised roots ( Lp _r) of young cucumber ( Cucumis sativus L.) seedlings as measured with the root pressure probe. A detailed analysis of root hydraulics provided evidence for a larger reduction in the osmotic component of Lp _r (77%) in comparison with the hydrostatic component (34%) in response to the exposure of the root system to 13°C. The activity of the plasma membrane H⁺-ATPase (EC 3.6.1.35) was reduced from 30 to 16 µmol Pi mg⁻¹ protein h⁻¹ upon exposure to 8°C for 1 day. Ultrastructural observations showed no evidence of loosening of the microstructure of endodermal cell walls in low temperature (LT)-treated roots. It is concluded that the rapid drop in the P _r in response to LT is largely caused by a reduction in the activity of the plasma membrane H⁺-ATPase rather than by loosening of the endodermal wall which would cause substantial solute losses. On the other hand, water permeability of root cell membrane at LT was related to changes in the activity (open/closed state) of water channels.     

Growth enhancement and antitranspirant activity following seed treatment with a derivative of 5-hydroxybenzimidazole (Ambiol) in four drought-stressed agricultural species

The biostimulating action of seed treatment with the synthetic antioxidant, Ambiol (2-methyl-4-Edimethylaminomethyl-5-hydroxybenzimidazole dihydrochloride) on subsequent growth and transpiration of seedlings was studied. To study growth and transpiration responses, seeds of four agricultural species, soybean ( Glycine max L.), rapeseed ( Brassica napus L.), winter wheat ( Trilicum aestivum L.) and corn ( Zea mays L.), were soaked in Ambiol for 24 h, using the following concentrations: 0, 0.01, 0.1, 1, 10 and 100 mg 1¹. The subsequent seedlings were subject to simulated soil drought, using computer-automated root misting chambers. The influence of Ambiot on transpiration rate under simulated air drought was studied by growing plants under low humidity in a controlled humidity chamber. Response to Ambiol varied, depending on its concentration, the species used and the environment. Compared to untreated plants, 10 mg 1-⁻¹ Ambiol reduced the mid-day transpiration rate and total daily water usage of soybean by approximately 25%. Under simulated soil drought in the root misting chamber, 10 and 100 mg 1-⁻¹ Ambiol increased growth of rapeseed and soybean by 25–45%, relative to the 0 mg 1-⁻¹ treatment, yielding plants comparable in size to the fully-irrigated controls. However, Ambiol failed to promote growth of two drought-stressed monocotyledons (corn and winter wheat). At 100 mg P. Ambiol inhibited growth of both well-watered wheat and rapeseed, although this inhibition was mitigated by drought.     

The effect of mechanical impedance on root growth in pea (Pisum sativum). I. Rates of cell flux, mitosis, and strain during recovery

We studied the effect of mechanical impedance on cell flux and meristematic activity in pea roots. Pea seedlings ( Pisum sativum L. cv. Helka) were grown in cores of sand packed to dry bulk densities of either; 1.4 Mg m⁻³ with an additional 2.4 kg uniaxial load applied to the surface to increase the mechanical resistance to growth (penetration resistance of 1.5 MPa); or 1.0 Mg m⁻³ (penetration resistance of 0.05 MPa). A water content of 0.06 g g⁻¹ was chosen for optimum root growth. After 3 days, the seedlings were transferred to hydroponics, colchicine was added and the rate of cell doubling, mitotic index and length of the cell cycle was assessed. Cell flux in the third cortical layer was calculated for roots immediately removed from sand.Mechanical impedance slowed root extension to about 20% of the unimpeded rate, and final cell length was reduced to 50% of the unimpeded length. The rate of cell doubling was 3.4 times slower for roots recovering from mechanical impedance mostly as a result of a longer period spent in interphase. Cell flux in impeded roots was approximately half that of unimpeded roots (5 cells h⁻¹), and contributed to a shorter cell file and elongation zone, and a slower rate of root elongation.     

Interaction of enriched CO2 and water stress on the physiology of and biomass production in sweet potato grown in open-top chambers

Abstract. The objective of this study was to investigate the effects of water stress in sweet potato ( Ipomoea batatas L. [Lam] 'Georgia Jet') on biomass production and plant-water relationships in an enriched CO₂ atmosphere. Plants were grown in pots containing sandy loam soil (Typic Paleudult) at two concentrations of elevated CO₂ and two water regimes in open-top field chambers. During the first 12 d of water stress, leaf xylem potentials were higher in plants grown in a CO₂ concentration of 438 and 666 μmol mol⁻¹ than in plants grown at 364 μmol mol⁻¹. The 364 μmol mol⁻¹ CO₂ grown plants had to be rewatered 2 d earlier than the high CO₂-grown plants in response to water stress. For plants grown under water stress, the yield of storage roots and root: shoot ratio were greater at high CO₂ than at 364 μmol mol⁻¹; the increase, however, was not linear with increasing CO₂ concentrations. In well-watered plants, biomass production and storage root yield increased at elevated CO₂, and these were greater as compared to water-stressed plants grown at the same CO₂ concentration.     

Antioxidant status of anoxia-tolerant and -intolerant plant species under anoxia and reaeration

The redox potential of the cell, as well as the antioxidant status of the tissue, are considered to be important regulatory constituents in an adaptive response in plants. Here the involvement of active antioxidants ascorbic acid (AA), reduced glutathione (GSH) and α - and β -tocopherols in reactive oxygen species scavenging, and the effect of anoxic stress on their reduction state were studied in 4 anoxia-tolerant and -intolerant plant species: Iris germanica L., Iris pseudacorus L., wheat ( Triticum aestivum L. cv. Leningradka) and rice ( Oryza sativa L. cv. VNIIR). The initial antioxidant content (both AA and GSH) was higher in the rhizomes of the more anoxia-tolerant Iris spp., as compared with that of the roots of the cereals. The predominant form of ascorbate was dehydroascorbic acid (DHA) in the cereals and AA in the Iris spp. Imposition of anoxia with subsequent reoxygenation resulted in an overall depletion of the reduced forms of antioxidants. No concurrent increase in oxidised forms (DHA and conjugated glutathione) was observed in anoxic samples. α -tocopherol content in Iris spp. was in the range 1–2 μg g⁻¹ fresh weight, while β -tocopherol content was higher in the anoxia-intolerant I. germanica (7.2 μg g⁻¹ fresh weight) as compared with the tolerant I. pseudacorus (1.5 μg g⁻¹ fresh weight). In I. pseudacorus , a significant decrease in α - and β -tocopherol levels was observed only after long-term (45 days) anoxia. The results suggested exclusion of AA and GSH from the redox cycling under prolonged anoxia, and a concomitant decrease in the redox state, as well as an anoxia-induced depletion of α - and β -tocopherols.     

Persistent xylem cross-walls reduce the axial hydraulic conductivity in the apical 20 cm of barley seminal root axes: implications for the driving force for water movement

Abstract. Xylem vessels in the apical 25 cm of barley seminal axes were examined by scanning electron microscopy of fractured freeze dried or critical point dried specimens. In the apical 11 cm, there were three cross walls cm⁻¹ root in the central xylem vessel. The frequency then declined with distance but did not become less than 1.0 cm⁻¹ root until the 22–25-cm zone.
Suction was applied to the proximal end of segments of seminal axes whose surfaces had been sealed with wax to prevent radial entry of water. Perfusion of the xylem with solutions of Tinopal CBS-X revealed the conductive xylem vessels by fluorescent staining of their walls. In the apical 20 cm of the axis, only a variable number of smaller xylem vessels conduct water. Beyond this zone, the much larger central vessel becomes functional.
The flow of water (Jv) in the apical zone was very much less for a given presure (△P) than in the proximal zone > 25 cm from the tip, and could be predicted by the Poiseuille equation provided the correct number of functional vessels are known. This information, together with earlier results on water uptake along the root length are used to predict the attenuation of the hydrostatic driving force for water uptake along the root length.
Estimates of K⁺ concentrations in stelar parenchyma and xylem vessels were made by electron microproble X-ray analysis. These results show that [K⁺] in the xylem vessels may be two to three times greater in the zone 1–2 cm from the root tip than in the basal zone. Such a gradient of solute potential may, to some extent, offset the decreasing influence of the leaf water potential in apical zones where xylem is not fully conductive.