Reactive oxygen species in the elongation zone of maize leaves are necessary for leaf extension

The production and role of reactive oxygen species (ROS) in the expanding zone of maize (Zea mays) leaf blades were investigated. ROS release along the leaf blade was evaluated by embedding intact seedlings in 2',7'-dichlorofluorescein-containing agar and examining the distribution of 2',7'-dichlorofluorescein fluorescence along leaf 4, which was exposed by removing the outer leaves before embedding the seedling. Fluorescence was high in the expanding region, becoming practically non-detectable beyond 65 mm from the ligule, indicating high ROS production in the expansion zone. Segments obtained from the elongation zone of leaf 4 were used to assess the role of ROS in leaf elongation. The distribution of cerium perhydroxide deposits in electron micrographs indicated hydrogen peroxide (H(2)O(2)) presence in the apoplast. 2',7'-Dichlorofluorescein fluorescence and apoplastic H(2)O(2) accumulation were inhibited with diphenyleneiodonium (DPI), which also inhibited O*(2)(-) generation, suggesting a flavin-containing enzyme activity such as NADPH oxidase was involved in ROS production. Segments from the elongation zone incubated in water grew 8% in 2 h. KI treatments, which scavenged H(2)O(2) but did not inhibit O*(2)(-) production, did not modify growth. DPI significantly inhibited segment elongation, and the addition of H(2)O(2) (50 or 500 microM) to the incubation medium partially reverted the inhibition caused by DPI. These results indicate that a certain concentration of H(2)O(2) is necessary for leaf elongation, but it could not be distinguished whether H(2)O(2), or other ROS, are the actual active agents.     

Salinity-induced decrease in NADPH oxidase activity in the maize leaf blade elongation zone

We reported previously that salinity-induced elongation constraints in the expansion zone of maize leaves are associated with reduced reactive oxygen species (ROS) production and could be alleviated by the addition of ROS. The NaCl effect was salt-specific and not osmotic. This paper explores the causes for such reduction. The decrease in ROS levels under salinity was not accompanied by increases in soluble apoplastic antioxidant activities such as superoxide dismutase, peroxidases and ascorbate. In experimental systems devoid of cell walls (protoplasts and membrane fractions) superoxide anion (O(2)(-)) production was inhibited by 50 and 100 mM NaCl, 50 microM DPI, 10 mM EGTA, and 5mM verapamil, a Ca(2+) channel inhibitor. Inhibitory effects of NaCl and reduced Ca(2+) supply were also observed in in gel assessment of O(2)(-) -generating activity. The main activity band excised from the ND-PAGE was recognized by an antibody against the C-terminal portion of the tomato gp91(phox) homolog. These results indicate the *O(2)(-) -generating activity negatively affected by NaCl was compatible with that of plasma membrane NADPH oxidase.     

Kinematics and Dynamics of Sorghum (Sorghum bicolor L.) Leaf Development at Various Na/Ca Salinities (I. Elongation Growth)

In many salt-sensitive species, elevated concentrations of Ca in the root growth media ameliorate part of the shoot growth reduction caused by NaCl stress. The physiological mechanisms by which Ca exerts protective effects on leaf growth are still not understood. Understanding growth inhibition caused by a stress necessitates locating the leaf expansion region and quantifying the profile of the growth reduction. This will enable comparisons and correlations with spatial gradients of probable physiologically inhibiting factors. In this work we applied the methods of growth kinematics to analyze the effects of elevated Ca concentrations on the spatial and temporal distributions of growth within the intercalary expanding region of salinized sorghum (Sorghum bicolor [L.] Moench, cv NK 265) leaves. NaCl (100 mM) caused a decrease in leaf elongation rate by shortening the leaf growing zone by 20%, as well as reducing the peak value of the longitudinal relative elemental growth rate (REG rate). Increasing the Ca concentrations from 1 to 10 mM restored the length of the growing zone of both emerged and unemerged salinized leaves and increased the peak value of the REG rate. The beneficial effects of supplemental Ca were, however, more pronounced in leaves after their appearance above the whorl of encircling older leaf sheaths. Elevated Ca then resulted in a peak value of REG rate higher than in the salinized leaves. The peak value of unemerged leaves was not increased, although it was maintained over a longer distance. The duration of elongation growth associated with a cell during its displacement from the leaf base was longer in salinized than control leaves, despite the fact that the elongation zone was shorter in salinity. Although partially restoring the length of the elongation region, supplemental Ca had no effect on the age of cessation of growth. Elongation of a tissue element, therefore, ceased when a cellular element reached a certain age and not a specific distance from the leaf base.     

Does salinity reduce growth in maize root epidermal cells by inhibiting their capacity for cell wall acidification?

The reduction in growth of maize (Zea mays L.) seedling primary roots induced by salinization of the nutrient medium with 100 millimolar NaCl was accompanied by reductions in the length of the root tip elongation zone, the length of fully elongated epidermal cells, and the apparent rate of cell production: Each was partially restored when calcium levels in the salinized growth medium were increased from 0.5 to 10.0 millimolar. We investigated the possibility that the inhibition of elongation growth by salinity might be associated with an inhibition of cell wall acidification, such as that which occurs when root growth is inhibited by IAA. A qualitative assay of root surface acidification, using bromocresol purple pH indicator in agar, showed that salinized roots, with and without extra calcium, produced a zone of surface acidification which was similar to that produced by control roots. The zone of acidification began 1 to 2 millimeters behind the tip and coincided with the zone of cell elongation. The remainder of the root alkalinized its surface. Kinetics of surface acidification were assayed quantitatively by placing a flat tipped pH electrode in contact with the elongation zone. The pH at the epidermal surfaces of roots grown either with 100 millimolar NaCl (growth inhibitory), or with 10 millimolar calcium ± NaCl (little growth inhibition), declined from 6.0 to 5.1 over 30 minutes. We conclude that NaCl did not inhibit growth by reducing the capacity of epidermal cells to acidify their walls.     

Rapid and reversible modifications of extension capacity of cell walls in elongating maize leaf tissues responding to root addition and removal of NaCl

A creep extensiometer technique was used to provide direct evidence that short (20 min) and long-term (3d) exposures of roots to growth inhibitory levels of salinity (100mol m^-3 NaCl) induce reductions in the irreversible extension capacity of cell walls in the leaf elongation zone of intact maize seedlings (Zea mays L.). The long-term inhibition of cell wall extension capacity was reversed within 20 min of salt withdrawal from the root medium. Inhibited elongation of leaf epidermal tissues was also reversed after salt removal. The salt-induced changes in wall extension capacity were detected using in vivo and in vitro assays (shortly after localized freeze/thaw treatment of the basal elongation zone). The rapid reversal of the inhibition of wall extensibility and leaf growth after salt removal from root medium of long-term salinized plants, suggested that neither deficiencies in growth essential mineral nutrients nor toxic effects of NaCl on plasmamembrane viability were directly involved in the inhibition of leaf growth. There was consistent agreement between the scale, direction and timing of salinity-induced changes in leaf elongation growth and wall extension capacity. Rapid metabolically regulated changes in the physical properties of growing cell walls, caused by osmotic (or other) effects, appear to be a factor regulating maize leaf growth responses to root salinization.     

Regulation of cell length in the Arabidopsis thaliana root by the ethylene precursor 1-aminocyclopropane- 1-carboxylic acid: a matter of apoplastic reactions

Treatment of the Arabidopsis thaliana root with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) immediately imposes a reduced maximal cell length beyond which further elongation is blocked. Here, we investigated possible apoplastic reactions involved in the inhibition of cell elongation. Five-day-old Arabidopsis seedlings were transferred to a growth medium supplemented with ACC and the effect on root cell length was recorded after 3 h of treatment. Altered characteristics in the apoplast of the nonelongating cells in the ACC-treated root, such as 'reactive oxygen species' (ROS) production and callose deposition, were detected using specific fluorochromes. The presence of functional hydroxyproline-rich glycoproteins (HRGPs) and the crosslinking of these cell-wall proteins are essential in limiting cell elongation. The ROS that drive the oxidative crosslinking of HRGPs, accumulate in the apoplast of cells in the zone where cell elongation stops. In the same cells, callose is deposited in the cell wall. The final cell length in the Arabidopsis root treated for a short period with ACC is determined in the zone of fast elongation. Both HRGPs crosslinking by ROS and callose deposition in the cell wall of this zone are suggested as causes for the reduced cell elongation.     

Effects of Salinity on the Extensibility and Ca Availability in the Expanding Region of Growing Barley Leaves

The growth of barley (Hordeum vulgare L.) leaves is reduced by salinity. We used the Instron extensometric technique to measure the reversible and irreversible compliance of the expanding regions of growing barley leaves from plants exposed to 1, 40, 80 and 120 mM NaCl in nutrient solution. Two barley cultivars differing in salinity resistance (cv ‘Arivat’ and cv ‘Briggs’) were compared over 5d of leaf growth. During the period of most active leaf expansion, salinity reduced reversible compliance and increased compliance in the leaf segments, although responses to salinity were complex and changed over the course of leaf expansion. Salinity increased irreversible compliance more in the salt-sensitive cultivar Arivat than in the more salt-tolerant cultivar Briggs. Elemental analysis of the basal leaf segments used for extensometry revealed an accumulation of Na and a depletion of Ca in segments from salinized plants, resulting in very high Na: Ca ratios in salinized expanding tissue. The concentrations of K and Mg in basal leaf tissue were elevated by salinity. Our data do support the hypothesis that the inhibition of leaf expansion by salinity stress is mediated by a decline in irreversible extensibility. We suggest that reduced Ca availability in expanding leaf tissue may contribute to growth reduction in salt-stressed barley seedlings.     

The biophysics of leaf growth in salt-stressed barley. A study at the cell level

Biophysical parameters potentially involved in growth regulation were studied at the single-cell level in the third leaf of barley (Hordeum vulgare) after exposure to various degrees of NaCl stress for 3 to 5 d. Gradients of elongation growth were measured, and turgor pressure, osmolality, and water potentials (psi) were determined (pressure probe and picoliter osmometry) in epidermal cells of the elongation zone and the mature blade. Cells in the elongation zone adjusted to decreasing external psi through increases in cell osmolality that were accomplished by increased solute loads and reduced water contents. Cell turgor changed only slightly. In contrast, decreases in turgor also contributed significantly to psi adjustment in the mature blade. Solute deposition rates in the elongation zone increased at moderate stress levels as compared with control conditions, but decreased again at more severe NaCl exposure. Growth-associated psi gradients between expanding epidermal cells and the xylem were significant under control and moderate stress conditions (75 mM NaCl) but seemed negligible at severe stress (120 mM NaCl). We conclude that leaf cell elongation in NaCl-treated barley is probably limited by the rate at which solutes can be taken up to generate turgor, particularly at high NaCl levels.     

Salinity-induced inhibition of leaf elongation in maize is not mediated by changes in cell wall acidification capacity

The physiological mechanisms underlying leaf growth inhibition under salt stress are not fully understood. Apoplastic pH is considered to play an important role in cell wall loosening and tissue growth and was demonstrated to be altered by several growth-limiting environmental conditions. In this study we have evaluated the possibility that inhibition of maize (Zea mays) leaf elongation by salinity is mediated by changes in growing cell wall acidification capacity. The kinetics of extended apoplast pH changes by leaf tissue of known expansion rates and extent of growth reduction under stress was investigated (in vivo) and was found similar for non-stressed and salt-stressed tissues at all examined apoplast salinity levels (0.1, 5, 10, or 25 mM NaCl). A similar rate of spontaneous acidification for the salt and control treatments was demonstrated also in in situ experiments. Unlike growing cells that acidified the external medium, mature nongrowing cells caused medium alkalinization. The kinetics of pH changes by mature tissue was also unchanged by salt stress. Fusicoccin, an enhancer of plasmalemma H(+)-ATPase activity level, greatly stimulated elongation growth and acidification rate to a similar extent in the control and salt treatments. That the ability of the growing tissue to acidify the apoplast did not change under same salt stress conditions that induced inhibition of tissue elongation rate suggests that salinity does not inhibit cell growth by impairing the acidification process or reducing the inherent capacity for cell wall acidification.     

Distribution of superoxide and hydrogen peroxide in Arabidopsis root and their influence on root development: possible interaction with peroxidases

The respective distribution of superoxide (O(2) (.-)) and hydrogen peroxide (H(2)O(2)), two reactive oxygen species (ROS) involved in root growth and differentiation, was determined within the Arabidopsis root tip. We investigated the effect of changing the levels of these ROS on root development and the possible interactions with peroxidases. H(2)O(2) was detected by confocal laser-scanning microscopy using hydroxyphenyl fluorescein (HPF). Both O(2) (.-) accumulation and peroxidase distribution were assessed by light microscopy, using nitroblue tetrazolium (NBT) and o-dianisidine, respectively. Root length and root hair length and density were also quantified following ROS scavenging. O(2) (.-) was predominantly located in the apoplast of cell elongation zone, whereas H(2)O(2) accumulated in the differentiation zone and the cell wall of root hairs in formation. Treatments that decrease O(2) (.-) concentration reduced root elongation and root hair formation, while scavenging H(2)O(2) promoted root elongation and suppressed root hair formation. The results allow to precise the respective role of O(2) (.-) and H(2)O(2) in root growth and development. The consequences of their distinct accumulation sites within the root tip are discussed, especially in relation to peroxidases.     

Apoplast Acidification in Growing Barley (Hordeum vulgare L.) Leaves

Apoplast acidification associated with growth is well documented in roots, coleoptiles, and internodes but not in leaves. In the present study, advantage was taken of the high cuticle permeability in the elongation zone of barley leaves to measure apoplast pH and growth in response to application of test reagents. The role of the plasma membrane H⁺-ATPase (PM-H⁺-ATPase) and K⁺ in this process was of particular interest. pH microelectrodes and an in vitro gel system with bromocresol purple as pH indicator were used to monitor apoplast pH. Growth was measured in parallel or in separate experiments using a linear variable differential transformer. Test reagents that blocked (vanadate) or stimulated (fusicoccin) PM-H⁺-ATPase or that reduced (Cs⁺, tetraethylammonium) K⁺ uptake were applied. Apoplast pH was lower in growing than in nongrowing leaf tissue and increased in the elongation zone with increasing apoplast K⁺. Vanadate increased apoplast pH and reduced growth, whereas fusicoccin caused the opposite effects. It is concluded that barley leaves exhibit acid-growth-type mechanisms in that apoplast pH is lower in elongating leaf tissue. Both growth and apoplast pH depend on the activity of the PM-H⁺-ATPase and K⁺ transport processes. However, not all of the growth displayed by leaves is dependent on a lower apoplast pH in the elongation zone; up to 50 % of growth is retained when apoplast pH in the elongation zone increases to a value observed in mature tissue.     

Axial changes in apoplast properties in the elongation zone of maize mesocotyl

Changes in extensibility of cell walls and composition of apoplastic solution along the elongation zone were studied in mesocotyls of etiolated seedlings of maize (Zea mays L.). It was found that plastic and elastic extensibility of cell walls was much greater in the cells with a high rate of elongation. Basipetal decrease in hydrogen peroxide concentration in the apoplast (from 5.1 to 2.0 ??M) was detected. We determined the activity of cell wall enzymes participating in H₂O₂ metabolism and found that in basal direction, potential ability of these enzymes to decompose H₂O₂ rises stronger than the ability to produce it. We found a basipetal decrease in polyamine oxidase activity, an increase in oxalate oxidase activity, and a rise in the ratio between peroxidase and NADH-oxidase activities of peroxidases. IAA (10^?6 M) promoted elongation of mesocotyl segments, induced a steady elevation of H₂O₂ content in the apoplast, an increase in NADH-oxidase activity of peroxidases, and a transient decrease in oxalate oxidase activity. Treatment with ABA (10^?4 M) suppressed elongation of mesocotyl segments, induced a transient elevation of H₂O₂ content in the apoplast, and a decrease in oxalate oxidase activity. It was shown that the main metabolites of apoplastic solution are glucose (20?C30 mM), fructose (6?C7 mM), malic acid (3 mM), and amino acids, namely, Asp, Glu, Asn, Gln, Ala, Val, Ser, Thr, and Phe. In basal direction, we observed a decrease in the content of glucose (from 30 to 20 mM), inositol (from 0.24 to 0.08 mM), and total amino acids (from 5.5 to 3.3 mM), whereas concentration of orthophosphate (3 mM) and malate (3 mM) did not change significantly. A relationship between the detected changes in the apoplast composition and basipetal decrease in the elongation rate of mesocotyl cells is discussed.     

Spatial and temporal quantitative analysis of cell division and elongation rate in growing wheat leaves under saline conditions

Leaf growth in grasses is determined by the cell division and elongation rates, with the duration of cell elongation being one of the processes that is the most sensitive to salinity. Our objective was to investigate the distribution profiles of cell production, cell length and the duration of cell elongation in the growing zone of the wheat leaf during the steady growth phase. Plants were grown in loamy soil with or without 120 mmol/L NaCl in a growth chamber, and harvested at day 3 after leaf 4 emerged. Results show that the elongation rate of leaf 4 was reduced by 120 mmol/L NaCl during the steady growth phase. The distribution profile of the lengths of abaxial epidermal cells of leaf 4 during the steady growth stage shows a sigmoidal pattern along the leaf axis for both treatments. Although salinity did not affect or even increased the length of the epidermal cells in some locations in the growth zone compared to the control treatment, the final length of the epidermal cells was reduced by 14% at 120 mmol/L NaCl. Thus, we concluded that the observed reduction in the leaf elongation rate derived in part from the reduced cell division rate and either the shortened cell elongation zone or shortened duration of cell elongation. This suggests that more attention should be paid to the effects of salinity on those properties of cell production and the period of cell maturation that are related to the properties of cell wall.     

Rapid and tissue-specific changes in ABA and in growth rate in response to salinity in barley leaves

The addition of 100 mM NaCl to the root medium of barley plantscaused the rapid cessation of elongation of the growing leafthree, followed by a sudden resumption of growth during thefollowing hour. The idea that resumption of growth is precededand mediated by rapid and tissue-specific changes in ABA concentrationand by changes in transpiration was tested. Leaf elongationvelocity was recorded continuously using linear variable displacementtransducers (LVDT), ABA was determined by immunoassay, and transpirationand stomatal conductivity were measured gravimetrically andby porometry, respectively. Within 10 min following additionof salt, ABA increased 6-fold in the distal portion of the leafelongation zone; in the proximal portion, ABA accumulated witha delay. In the portion of the growing blade that had emergedABA increased 3-fold and remained elevated during the following20 min. This preceded a decrease in transpiration and stomatalconductivity, which, in turn, coincided with growth resumption.Twenty hours following the addition of salt, the ABA concentrationshad returned to the level before stress. Leaf elongation velocitywas still reduced. It is concluded that NaCl causes a rapidincrease in ABA in the transpiring portion of the growing leaf.This leads to a decrease in transpiration. As a result, xylemwater potential is expected to rise. The moment that the waterpotential gradient between the xylem and the peripheral cellsin the growth zone favours water uptake again into the latter,leaf elongation resumes. The results suggest that ABA causesdifferent responses in different leaf regions, all aimed atpromoting the resumption of leaf growth. Key words: Abscisic acid, cell elongation, Hordeum vulgare, leaf growth, salinity, water relations.     

Leaf gas exchange and solute accumulation in the halophyte Salvadora persica grown at moderate salinity

The domestication of halophytes has been proposed as a strategy to expand cultivation onto unfavorable land. However, halophytes mainly have been considered for their performance in extremely saline environments, and only a few species have been characterized in terms of their tolerance and physiological responses to moderately high levels of salinity. Salvadora persica is an evergreen perennial halophyte capable of growing under extreme conditions, from very dry environments to highly saline soils. It possesses high potential economic value as a source of oil and medicinal compounds. To quantify its response to salinity, S. persica seedlings were exposed to 200 mM NaCl for 3 weeks, and growth, leaf gas exchange and solute accumulation were measured. The presence of NaCl induced a 100% increase in fresh weight and a 30% increase in dry weight, relative to non-salinized controls. Increases in fresh weight and dry weight were not associated with higher rates of net CO(2) assimilation, however. Analysis of ion accumulation revealed that S. persica leaves accumulated Na(+) as a primary osmoticum. The concentration of Na(+) in leaves of salinized plants was approximately 40-fold greater than that measured in non-salinized controls, and this was associated with significant reductions in leaf K(+) and Ca(2+) concentrations. In addition, a significant accumulation of proline, probably associated with osmotic adjustment and protection of membrane stability, occurred in roots of salinized plants.     

Salinity effects on polyphenol content and antioxidant activities in leaves of the halophyte Cakile maritima.

Cakile maritima is a local oilseed halophyte exhibiting potential for secondary metabolite production. In the present study, plant growth, leaf polyphenol content and antioxidant activity were comparatively analyzed in two C. maritima Tunisian accessions (Jerba and Tabarka, respectively sampled from arid and humid bioclimatic stages) under salt constraint. Three-week-old plants were subjected to 0, 100, and 400 mM NaCl for 28 days under glasshouse conditions. A significant variability in salt response was found between both accessions: while Tabarka growth (shoot biomass, leaf expansion) was significantly restricted at 100 and 400 mM NaCl, compared to the control, Jerba growth increased at 100mM before declining at 400 mM NaCl. The better behaviour of Jerba salt-challenged plants, compared to those of Tabarka, may be related to their higher polyphenol content (1.56- and 1.3-fold the control, at 100 and 400 mM NaCl respectively) and antioxidant activity (smaller IC(50) values for both 1,1-diphenyl-2-picrylhydrazyl and superoxide scavenging), associated with lower leaf MDA accumulation (ca. -66% of the control at 100mM NaCl). Taken together, our findings suggest that halophytes may be interesting for production of antioxidant compounds, and that the accession-dependent capacity to induce antioxidative mechanisms in response to salt, may result in a corresponding variability for growth sustainability.     

Growth performance and physiological response in the halophyte Lycium barbarum grown at salt-affected soil

As a traditional Chinese medicinal plant, Lyciumbarbarum is of high economic value and has attracted many considerable interests in recent years. The plant is a perennial halophyte grown under extreme conditions, especially under highly saline soil. A pot experiment was carried out to quantify the responses of L. barbarum plants to soil salinity applied at 100 and 200 mM NaCl. The results demonstrate that 100 mM NaCl soil improves the growth of L. barbarum seedlings. Because the 100 mM NaCl soil enhanced plant height and dry matter by 20% and 30% compared with the nonsalinised soil, it is considered suitable, and the 200 mM NaCl soil showed negative effects, too extreme for the growth of L. barbarum. The leaf cations and betaine content increased significantly under salt stress. The leaf chlorophyll, gas exchange, photochemical efficiency, leaf area and soluble sugar contents showed a significant decrease under 200 mM NaCl stress compared with the nonsalinised and the 100 mM NaCl‐affected soil. The results do not provide a basic mechanism for the observed growth stimulation; however, they suggest that L. barbarum may be an economic species for cultivation in moderately saline areas such as northwest China.     

Involvement of kinetin and spermidine in controlling salinity stress in mulberry (Morus alba L. cv. S1)

In mulberry (Morus alba L.) plants NaCl stress imposed through roots by irrigation during growth period decreased the net photosynthetic rate (NPR), physiological water use efficiency (pWUE), which ultimately reflected on the reduction of growth parameters and leaf yield. Foliar spray of kinetin and spermidine (both at 1 mM) on salinized plants reduced the detrimental effects of saline stress. Kinetin and spermidine sprayed plants increased the total chlorophyll, protein content, as well as leaf yield, but reduced the sugar and proline contents as compared to NaCl treated plants. Kinetin was more effective than spermidine in increasing NPR, pWUE and leaf yield both in nonsalinized and salinized condition.