The spatially variable inhibition by water deficit of maize root growth correlates with altered profiles of proton flux and cell wall pH

Growth of elongating primary roots of maize (Zea mays) seedlings was approximately 50% inhibited after 48 h in aerated nutrient solution under water deficit induced by polyethylene glycol 6000 at -0.5 MPa water potential. Proton flux along the root elongation zone was assayed by high resolution analyses of images of acid diffusion around roots contacted for 5 min with pH indicator gel. Profiles of root segmental elongation correlated qualitatively and quantitatively (r(2) = 0.74) with proton flux along the surface of the elongation zone from water-deficit and control treatments. Proton flux and segmental elongation in roots under water deficit were remarkably well maintained in the region 0 to 3 mm behind the root tip and were inhibited from 3 to 10 mm behind the tip. Associated changes in apoplastic pH inside epidermal cell walls were measured in three defined regions along the root elongation zone by confocal laser scanning microscopy using a ratiometric method. Finally, external acidification of roots was shown to specifically induce a partial reversal of growth inhibition by water deficit in the central region of the elongation zone. These new findings, plus evidence in the literature concerning increases induced by acid pH in wall-extensibility parameters, lead us to propose that the apparently adaptive maintenance of growth 0 to 3 mm behind the tip in maize primary roots under water deficit and the associated inhibition of growth further behind the tip are related to spatially variable changes in proton pumping into expanding cell walls.     

Auxin-induced changes in cell wall extensibility of maize roots

The rheological properties of corn (Zea mays L. cv. Garant) root elongation zones were investigated by means of a computer-controlled extensiometer. Creep closely followed a logarithmic time function, which was used to quantify creep activity. Pretreatment with auxin, which inhibits extension growth in roots, lowered the creep activity and the apparent plastic extensibility. While the time course of the inhibition of apparent plastic extensibility lagged behind the cessation of elongation growth, the drop in creep activity matched the growth inhibition more closely. Creep activity and apparent plastic extensibility were not significantly affected by pH. These data support the view that the auxin-induced cell wall stiffening (e.g. by cross-linking processes), while causal for the growth inhibition, is not brought about by a cell wall alkalinization. Received: 10 December 1996 / Accepted: 19 August 1997     

Cadmium induces premature xylogenesis in barley roots

The effect of Cd on H₂O₂ production, peroxidase (POD) activity and root hair formation were analyzed in barley root. Cd causes a strong H₂O₂ burst in the root region 0–6 mm behind the root tip. POD activity was activated in root tip and raised toward the root base in Cd treated roots. In situ analyses showed that both elevated H₂O₂ production and POD activity are localized in the early metaxylem vascular bundles. Cd induces root hair formation in the region 2 to 4 mm behind the root tip that was not detected in control roots. These results suggest that Cd-induced root growth inhibition is at least partially the consequence of Cd-stimulated premature root development involving xylogenesis and root hair formation, which is correlated with shortening of root elongation zone and therefore with root growth reduction.     

Separate de Novo and Salvage Purine Pools Are Involved in the Biosynthesis of Theobromine but Not Caffeine in Leaves of Coffea arabica L

We used a video digitizer system to (a) measure changes in the pattern of longitudinal surface extension in primary roots of maize (Zea mays L.) upon application and withdrawal of auxin and (b) compare these patterns during gravitropism in control roots and roots pretreated with auxin. Special attention was paid to the distal elongation zone (DEZ), arbitrarily defined as the region between the meristem and the point within the elongation zone at which the rate of elongation reaches 0.3 of the peak rate. For roots in aqueous solution, the basal limit of the DEZ is about 2.5 mm behind the tip of the root cap. Auxin suppressed elongation throughout the elongation zone, but, after 1 to 3 h, elongation resumed, primarily as a result of induction of rapid elongation in the DEZ. Withdrawal of auxin during the period of strong inhibition resulted in exceptionally rapid elongation attributable to the initiation of rapid elongation in the DEZ plus recovery in the main elongation zone. Gravistimulation of auxin-inhibited roots induced rapid elongation in the DEZ along the top of the root. This resulted in rapid gravitropism even though the elongation rate of the root was zero before gravistimulation. The results indicate that cells of the DEZ differ from cells in the bulk of the elongation zone with respect to auxin sensitivity and that DEZ cells play an important role in gravitropism.     

Lignin deposition induced by aluminum in wheat (Triticum aestivum) roots

We investigated the relation between the toxic effect of aluminum (Al) on root growth and the lignin deposition in wheat ( Triticum aestivum L. cvs Atlas 66 and Scout 66). In the Al-tolerant cultivar Atlas 66, control treatment without AlCl₃ at pH 4.75, cell length increased dramatically in the portion of the root that was 0.6 to 3.2 mm from the root cap junction (approximately 1.0 to 3.6 mm from the root tip). However, treatment with 20 μ M AlCl₃ for 24 and 48 h completely inhibited root elongation and markedly decreased the length and increased the diameter of the cells in the same portion of the root. Moreover, marked deposition of lignin was observed in the cells that corresponded to the portion 1.5 to 4.5 mm from the root tip in Atlas 66 roots treated with 20 μ M AlCl₃, while no deposition of lignin was detected in control roots. Treatment with 5 μ M AlCl₃ slightly inhibited root growth and there was no deposition of lignin in the root. On the other hand, in roots of the Al-sensitive cultivar Scout 66, treatment with 5 μ M AlCl₃ completely inhibited root growth and markedly induced deposition of lignin. These results suggest that lignification in the elongating region coincided with the extent of inhibition of root growth by Al in two wheat cultivars that differed in their sensitivity to Al.     

Impact of the auxin signaling inhibitor p-chlorophenoxyisobutyric acid on short-term Cd-induced hydrogen peroxide production and growth response in barley root tip

Short-term treatment (30min) of barley roots with a low 10μM Cd concentration induced significant H(2)O(2) production in the elongation and differentiation zone of the root tip 3h after treatment. This elevated H(2)O(2) production was accompanied by root growth inhibition and probably invoked root swelling in the elongation zone of the root tip. By contrast, a high 60μM Cd concentration induced robust H(2)O(2) production in the elongation zone of the root tip already 1h after short-term treatment. This robust H(2)O(2) generation caused extensive cell death 6h after short-term treatment. Similarly to low Cd concentration, exogenously applied H(2)O(2) caused marked root growth inhibition, which at lower H(2)O(2) concentration was accompanied by root swelling. The auxin signaling inhibitor p-chlorophenoxyisobutyric acid effectively inhibited 10μM Cd-induced root growth inhibition, H(2)O(2) production and root swelling, but was ineffective in the alleviation of 60μM Cd-induced root growth inhibition and H(2)O(2) production. Our results demonstrated that Cd-induced mild oxidative stress caused root growth inhibition, likely trough the rapid reorientation of cell growth in which a crucial role was played by IAA signaling in the root tip. Strong oxidative stress induced by high Cd concentration caused extensive cell death in the elongation zone of the root tip, resulting in the cessation of root growth or even in root death.     

Hardening of root cell walls: a growth inhibitory response to salinity stress

Primary roots of intact maize plants (Zea mays L.) grown for several days in nutrient solutions containing 100 mol m⁻³ NaCl and additional calcium, had relatively inhibited rates of elongation. Possible physical restraints underlying this salt induced inhibition were investigated. The inhibition did not involve reductions in osmotic potential gradients and turgor in the tip tissues responsible for root elongation growth. The apparent yield threshold pressure, which is related to capacity of cell walls to undergo loosening by stress relaxation, was estimated psychrometrically in excised root tips. Salinity increased yield threshold values. Comparative root extensibility values were obtained for intact plants by determining the initial (1 min) increase in root elongation rate induced by an 0.1 MPa osmotic jump. Comparative extensibility was significantly reduced in the salinized root tips. Salinity did not reduce capacities for water efflux and associated elastic contraction in root tip tissues of intact plants exposed to hypertonic mannitol. We conclude that cell wall hardening in the elongating root tips is an important component of root growth inhibition induced by long-term salinization.     

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.     

Aluminum-Induced Rapid Root Inhibition and Changes in Cell-Wall Components of Squash Seedlings

Growth of squash (Cucurbita maxima Duch.) roots was significantly inhibited by 1 mM AlCl3 as early as 1 h after the treatment. The growth inhibition was confined to the elongating zone (1-6 mm from the root tip). Chemical analysis of cell-wall polysaccharides from roots revealed that aluminum increased pectin, hemi-cellulose, and cellulose contents after 3 h of treatment. The effect of aluminum on pectin content was found in the elongating zone including the root tip, whereas change in cellulose content was confined to only nonelongating zones. Hemicellulose content increased in all of the regions along the root axis. The increase in the pectin fraction was due to the increases in uronic acids, galactose, and arabinose constituents, whereas hemicellulose content changed due to increases in glucose, xylose, galactose, and arabinose. The results clearly indicate that aluminum rapidly reduced squash root growth by inhibiting cell elongation and altering metabolism of cell-wall polysaccharides in the nonelongating zone as well as in the elongating zone.     

Morphological changes in the apex of pea roots during and after recovery from aluminium treatment

We investigated how the pea (Pisum sativum cv. Harunoka) root, upon return to an Al-free condition, recovers from injury caused by exposure to Al. The growing region of the root during and after treatment with Al was examined by marking the root at intervals with India ink. Al-induced cell death was detected by staining with Evans blue. Root growth in 40 μM Al solution relative to that in Al-free solution (RRG) was approximately 45% from 6 h to12 h after the start of the treatment. However, values of RRG from 12 h to 24 h in Al-free solution for recovery or in the same Al solution were about 75% and 35%, respectively, indicating recovery from Al-induced growth inhibition. Images of the root characterized by zonal staining with Evans blue were observed in the sub-apical region (more than 1 mm from the tip) in Al-stressed roots. However, the interval of the stained zone was widened in the root after recovery from Al-induced growth inhibition, though it was narrower and more densely stained with time in the Al-stressed roots. During the recovery, the root apex may resume elongation in a specified region without Al-induced death or injury in cells detected by Evans blue.     

Relation of growth process to spatial patterns of electric potential and enzyme activity in bean roots

The electric spatial pattern and invertase activity distribution in growing roots of azuki bean (Phaseolus chrysanthos) have been studied. The electric potential near the surface along the root showed a banding pattern with a spatial period of about 2 cm. It was found that the enzyme activity has a peak around 3-7 mm from the root tip, in good agreement with the position of the first peak of the electric potential, which is located a little behind the elongation zone. An inhomogeneous distribution of ATP content was also detected along the root. Experiments on the electric isolation of the elongation zone from the mature zone and acidification treatment showed that H+ is transported from the mature-side to elongation-side regions, causing tip elongation through an acid-growth mechanism. Both acidification and electric disturbance on growing roots affected growth significantly. Simultaneous measurements of electric potential and enzyme activity clearly showed a good correlation between these two quantities and growth speed. From an analogy with the Characean banding, the spatio-temporal organization via the cell membrane in electric potential and enzyme activity can be regarded as a dissipative structure arising far from equilibrium. These experimental results can be interpreted with a new mechanism that the dissipative structure is formed spontaneously along the whole root, accompanied by energy metabolism, to make H+ flow into the root tip.     

Differential expression of cell wall related genes in the elongation zone of rice roots under water deficit

Growth in the apical elongation zone of plant roots is central to the development of functional root systems. It has been known that rice seminal root elongation could be enhanced by water stress. In the present study, 17 cell-wall related genes were identified by cDNA-amplified fragment length polymorphism (cDNA-AFLP) technique. Five genes encoded cell-wall loosening enzymes and six genes were involved in the lignin biosynthesis. The six other genes were related to the metabolism of polysaccharide and protein matrices in cell wall. Northern blot analysis confirmed that they were differentially expressed in the elongation zone of rice seminal roots under water stress, and none of them was root-specific. The results indicated that the activity of cell-wall loosening enzymes was enhanced in the early stage (within 16 h), and some cell wall matrices were synthesized rapidly in the middle stages (from 16 to 48 h), while lignin biosynthesis was enhanced in the middle and late stages of water stress (from 48 to 72 h). Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 3, pp. 437–443. The text was submitted by the authors in English.     

Cell wall proteome in the maize primary root elongation zone. II. Region-specific changes in water soluble and lightly ionically bound proteins under water deficit

Previous work on the adaptation of maize (Zea mays) primary roots to water deficit showed that cell elongation is maintained preferentially toward the apex, and that this response involves modification of cell wall extension properties. To gain a comprehensive understanding of how cell wall protein (CWP) composition changes in association with the differential growth responses to water deficit in different regions of the elongation zone, a proteomics approach was used to examine water soluble and loosely ionically bound CWPs. The results revealed major and predominantly region-specific changes in protein profiles between well-watered and water-stressed roots. In total, 152 water deficit-responsive proteins were identified and categorized into five groups based on their potential function in the cell wall: reactive oxygen species (ROS) metabolism, defense and detoxification, hydrolases, carbohydrate metabolism, and other/unknown. The results indicate that stress-induced changes in CWPs involve multiple processes that are likely to regulate the response of cell elongation. In particular, the changes in protein abundance related to ROS metabolism predicted an increase in apoplastic ROS production in the apical region of the elongation zone of water-stressed roots. This was verified by quantification of hydrogen peroxide content in extracted apoplastic fluid and by in situ imaging of apoplastic ROS levels. This response could contribute directly to the enhancement of wall loosening in this region. This large-scale proteomic analysis provides novel insights into the complexity of mechanisms that regulate root growth under water deficit conditions and highlights the spatial differences in CWP composition in the root elongation zone.     

Involvement of nitric oxide in water stress-induced responses of cucumber roots

The effect of water deficit on nitric oxide (NO) generation was investigated in cucumber (Cucumis sativus cv. Dar) seedling roots using bio-imaging with an NO-selective fluorophor, diaminofluorescein-2-diacetate (DAF-2DA). Roots subjected to mild (5 and 10 h) water deficit showed slightly enhanced NO synthesis in cells of root tips and in the surrounding elongation zone. However, severe (17 h) stress resulted in an intensive NO production localized mainly in and above the elongation zone. Water stress-induced NO generation was blocked by a specific NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) as well as nitrate reductase (NR) and partially by nitric oxide synthase (NOS-like) inhibitors.A pharmacological approach was used in order to verify the capacity of NO to induce adaptive responses of cucumber roots to water deficit. A positive correlation was found between NO donor (SNP 100 μM and GSNO 100 μM) pretreatment and plant hydration status, measured as relative water content (RWC) during progressive dehydration. At an early stage (5 h) of stress duration NO caused a periodical increase in lipoxygenase (LOX) activity, correlated with time-dependent enhancement of lipid peroxidation. Beginning from 10 h up to severe stress (17 h) exogenous NO was able to diminish LOX activity and alleviate water deficit-induced membrane permeability and lipid peroxidation, measured as TBARS content and visualised by histochemical staining in situ. Observed changes via NO were accompanied by a significant reduction of proline level, suggesting that the accumulation of this osmolyte might not be essential in water stress tolerance. Obtained results clearly indicate that NO augmentation is likely to help the plant at the initial stage of tissue dehydration to trigger efficient mechanisms, mitigating severe water deficit effects in roots of cucumber seedlings.     

Effect of partial root excision on shoot water relations

Removing 4 out of 5 serminal roots from 7-day-old wheat seedlings arrested leaf elongation for 1.5 h. This effect can be explained by an initial decrease in foliar water content resulting from the smaller root surface area available for water uptake. Subsequently, leaf hydration increased with time and came to equal that of intact plants within 2 h. The rehydration was seemingly effected by an increasing conductivity of the one remaining root axis, since transpiration of the partially de-rooted plants did not fall below that of controls. With time, leaf elongation resumed, but at a slower rate than in intact plants. This slower growth may be attributed to a decrease in leaf extensibility since this was found to be reduced when measured by a counterweight technique involving linear displacement transducers. Loss of extensibility was associated with decreased IAA concentration in the leaf elongation zone.     

Band structure of surface electric potential in growing roots

For growing roots of azuki bean (Phaseolus chrysanthos), an electric potential is measured minutely along the surface of the root, together with the surface pH. It was found that the root begins to display a band-type pattern of potential with a spatial period of about 2 cm in a mature region as soon as it grows to about 10 cm in root length, while the surface potential shows only one convex peak around a position 5-20 mm behind a root tip and a succeeding concave peak around 20-35 mm, providing the length of root is shorter than about 10 cm. Since the surface potential takes a relatively positive value on average at the side of the root base compared with that in an elongation zone near the tip, electric current is supposed to flow into the elongation zone, accompanied by some local current loops in the mature region. The present band-type pattern observed first in multi-cell systems seems to be a kind of dissipative structure appearing far from equilibrium, and hence its relationship to growth is discussed.     

Growth dynamics of mechanically impeded lupin roots: does altered morphology induce hypoxia?

BACKGROUND AND AIMS: Root axes elongate slowly and swell radially under mechanical impedance. However, temporal and spatial changes to impeded root apices have only been described qualitatively. This paper aims (a) to quantify morphological changes to root apices and (b) assess whether these changes pre-dispose young root tissues to hypoxia. METHODS: Lupin (Lupinus angustifolius) seedlings were grown into coarse sand that was pressurized through a diaphragm to generate mechanical impedance on growing root axes. In situ observations yielded growth rates and root response to hypoxia. Roots were then removed to assess morphology, cell lengths and local growth velocities. Oxygen uptake into excised segments was measured. KEY RESULTS: An applied pressure of 15 kPa slowed root extension by 75% after 10-20 h while the same axes thickened by about 50%. The most terminal 2-3 mm of axes did not respond morphologically to impedance, in spite of the slower flux of cells out of this region. The basal boundary of root extension encroached to within 4 mm of the apex (cf. 10 mm in unimpeded roots), while radial swelling extended 10 mm behind the apex in impeded roots. Oxygen demand by segments of these short, thick, impeded roots was significantly different from segments of unimpeded roots when the zones of elongation in each treatment were compared. Specifically, impeded roots consumed O2 faster and O2 consumption was more likely to be O2-limited over a substantial proportion of the elongation zone, making these roots more susceptible to O2 deficit. Impeded roots used more O2 per unit growth (measured as either unit of elongation or unit of volumetric expansion) than unimpeded roots. Extension of impeded roots in situ was O2-limited at sub-atmospheric O2 levels (21% O2), while unimpeded roots were only limited below 11% O2. CONCLUSIONS: The shift in the zone of extension towards the apex in impeded roots coincided with greater vulnerability to hypoxia even after soil was removed. Roots still encased in impeded soil are likely to suffer from marked O2 deficits.     

WEG1, which encodes a cell wall hydroxyproline-rich glycoprotein,is essential for parental root elongation controlling lateral root formation in rice

Lateral roots (LRs) determine the overall root system architecture, thus enabling plants to efficiently explore their underground environment for water and nutrients. However, the mechanisms regulating LR development are poorly understood in monocotyledonous plants. We characterized a rice mutant, wavy root elongation growth 1 (weg1), that produced higher number of long and thick LRs (L-type LRs) formed from the curvatures of its wavy parental roots caused by asymmetric cell growth in the elongation zone. Consistent with this phenotype, was the expression of the WEG1 gene, which encodes a putative member of the hydroxyproline-rich glycoprotein family that regulates cell wall extensibility, in the root elongation zone. The asymmetric elongation growth in roots is well known to be regulated by auxin, but we found that the distribution of auxin at the apical region of the mutant and the wild-type roots was symmetric suggesting that the wavy root phenotype in rice is independent of auxin. However, the accumulation of auxin at the convex side of the curvatures, the site of L-type LR formation, suggested that auxin likely induced the formation of L-type LRs. This was supported by the need of a high amount of exogenous auxin to induce the formation of L-type LRs. These results suggest that the MNU-induced weg1 mutated gene regulates the auxin-independent parental root elongation that controls the number of likely auxin-induced L-type LRs, thus reflecting its importance in improving rice root architecture.