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.     

Cell wall integrity controls root elongation via a general 1-aminocyclopropane-1-carboxylic acid-dependent, ethylene-independent pathway

Cell expansion in plants requires cell wall biosynthesis and rearrangement. During periods of rapid elongation, such as during the growth of etiolated hypocotyls and primary root tips, cells respond dramatically to perturbation of either of these processes. There is growing evidence that this response is initiated by a cell wall integrity-sensing mechanism and dedicated signaling pathway rather than being an inevitable consequence of lost structural integrity. However, the existence of such a pathway in root tissue and its function in a broader developmental context have remained largely unknown. Here, we show that various types of cell wall stress rapidly reduce primary root elongation in Arabidopsis (Arabidopsis thaliana). This response depended on the biosynthesis of 1-aminocyclopropane-1-carboxylic acid (ACC). In agreement with the established ethylene signaling pathway in roots, auxin signaling and superoxide production are required downstream of ACC to reduce elongation. However, this cell wall stress response unexpectedly does not depend on the perception of ethylene. We show that the short-term effect of ACC on roots is partially independent of its conversion to ethylene or ethylene signaling and that this ACC-dependent pathway is also responsible for the rapid reduction of root elongation in response to pathogen-associated molecular patterns. This acute response to internal and external stress thus represents a novel, noncanonical signaling function of ACC.     

Changes in root cap pH are required for the gravity response of the Arabidopsis root

Although the columella cells of the root cap have been identified as the site of gravity perception, the cellular events that mediate gravity signaling remain poorly understood. To determine if cytoplasmic and/or wall pH mediates the initial stages of root gravitropism, we combined a novel cell wall pH sensor (a cellulose binding domain peptide-Oregon green conjugate) and a cytoplasmic pH sensor (plants expressing pH-sensitive green fluorescent protein) to monitor pH dynamics throughout the graviresponding Arabidopsis root. The root cap apoplast acidified from pH 5.5 to 4.5 within 2 min of gravistimulation. Concomitantly, cytoplasmic pH increased in columella cells from 7.2 to 7.6 but was unchanged elsewhere in the root. These changes in cap pH preceded detectable tropic growth or growth-related pH changes in the elongation zone cell wall by 10 min. Altering the gravity-related columella cytoplasmic pH shift with caged protons delayed the gravitropic response. Together, these results suggest that alterations in root cap pH likely are involved in the initial events that mediate root gravity perception or signal transduction.     

Apoplastic alkalinization is instrumental for the inhibition of cell elongation in the Arabidopsis root by the ethylene precursor 1-aminocyclopropane-1-carboxylic acid

In Arabidopsis (Arabidopsis thaliana; Columbia-0) roots, the so-called zone of cell elongation comprises two clearly different domains: the transition zone, a postmeristematic region (approximately 200-450 μm proximal of the root tip) with a low rate of elongation, and a fast elongation zone, the adjacent proximal region (450 μm away from the root tip up to the first root hair) with a high rate of elongation. In this study, the surface pH was measured in both zones using the microelectrode ion flux estimation technique. The surface pH is highest in the apical part of the transition zone and is lowest at the basal part of the fast elongation zone. Fast cell elongation is inhibited within minutes by the ethylene precursor 1-aminocyclopropane-1-carboxylic acid; concomitantly, apoplastic alkalinization occurs in the affected root zone. Fusicoccin, an activator of the plasma membrane H(+)-ATPase, can partially rescue this inhibition of cell elongation, whereas the inhibitor N,N'-dicyclohexylcarbodiimide does not further reduce the maximal cell length. Microelectrode ion flux estimation experiments with auxin mutants lead to the final conclusion that control of the activity state of plasma membrane H(+)-ATPases is one of the mechanisms by which ethylene, via auxin, affects the final cell length in the root.     

Cell wall pectic (1-->4)-beta-d-galactan marks the acceleration of cell elongation in the Arabidopsis seedling root meristem

Here we demonstrate that the pectic rhamnogalacturonan-I-associated LM5 (1-->4)-beta-d-galactan epitope occurs in a restricted manner at the root surface of intact Arabidopsis seedlings. The root surface occurrence of (1-->4)-beta-d-galactan marks the transition zone at or near the onset of rapid cell elongation and the epitope is similarly restricted in occurrence in epidermal, cortical and endodermal cell walls. The extent of surface (1-->4)-beta-d-galactan occurrence is reduced in response to genetic mutations (stp-1, ctr-1) and hormone applications that reduce root cell elongation. In contrast, the application of the arabinogalactan-protein (AGP) binding beta-glucosyl Yariv reagent (betaGlcY) that disrupts cell elongation results in the persistence of (1-->4)-beta-d-galactan at the root surface and in epidermal, cortical and endodermal cell walls. This latter observation indicates that modulation of pectic (1-->4)-beta-d-galactan may be an event downstream of AGP function during cell expansion in the Arabidopsis seedling root.     

Position and cell type-dependent microtubule reorientation characterizes the early response of the Arabidopsis root epidermis to ethylene

The involvement of cortical microtubules in the control of plant cell expansion was studied in the Arabidopsis root epidermis. In the zone of fast elongation microtubules were transverse to the root axis in all epidermal cells. However when cells entered the differentiation zone cell type-specific microtubule reorientation took place. In the trichoblasts that were then approximately 130 µm long and formed the root hair bulge, the microtubules switched to a random distribution. In the adjoining atrichoblasts microtubules adopted a slightly oblique orientation. In more proximal parts of the differentiation zone atrichoblast microtubules were found in a more oblique and finally in a longitudinal orientation. Upon exposure to ethylene or 1-aminocyclopropane-1-carboxylic acid (ACC – the precursor of ethylene) at a saturating dose, cell elongation abruptly stopped. From then on trichoblast cells reached only a length of about 35 µm, and developed root hairs. Cortical microtubules changed orientation within 10 min. In trichoblasts they adopted the typical random orientation, in atrichoblasts however, they took up a longitudinal orientation. Microtubule reorientation was complete within 60 min. The possible role of microtubules in the control of cell elongation is discussed.     

Expression of AtPRP3, a proline-rich structural cell wall protein from Arabidopsis, is regulated by cell-type-specific developmental pathways involved in root hair formation

The tightly regulated expression patterns of structural cell wall proteins in several plant species indicate that they play a crucial role in determining the extracellular matrix structure for specific cell types. We demonstrate that AtPRP3, a proline-rich cell wall protein in Arabidopsis, is expressed in root-hair-bearing epidermal cells at the root/shoot junction and within the root differentiation zone of light-grown seedlings. Several lines of evidence support a direct relationship between AtPRP3 expression and root hair development. AtPRP3/beta-glucuronidase (GUS) expression increased in roots of transgenic seedlings treated with either 1-aminocyclopropane-1-carboxylic acid (ACC) or alpha-naphthaleneacetic acid (alpha-NAA), compounds known to promote root hair formation. In the presence of 1-alpha-(2-aminoethoxyvinyl)glycine (AVG), an inhibitor of ethylene biosynthesis, AtPRP3/GUS expression was strongly reduced, but could be rescued by co-addition of ACC or alpha-NAA to the growth medium. In addition, AtPRP3/GUS activity was enhanced in ttg and gl2 mutant backgrounds that exhibit ectopic root hairs, but was reduced in rhd6 and 35S-R root-hair-less mutant seedlings. These results indicate that AtPRP3 is regulated by developmental pathways involved in root hair formation, and are consistent with AtPRP3's contributing to cell wall structure in Arabidopsis root hairs.     

Decreased reactive oxygen species concentration in the elongation zone contributes to the reduction in maize leaf growth under salinity

Reactive oxygen species (ROS) in the apoplast of cells in the growing zone of grass leaves are required for elongation growth. This work evaluates whether salinity-induced reductions in leaf elongation are related to altered ROS production. Studies were performed in actively growing segments (SEZ) obtained from leaf three of 14-d-old maize (Zea mays L.) seedlings gradually salinized to 150 mM NaCl. Salinity reduced elongation rates and the length of the leaf growth zone. When SEZ obtained from the elongation zone of salinized plants (SEZs) were incubated in 100 mM NaCl, the concentration where growth inhibition was approximately 50%, O2*- production, measured as NBT formazan staining, was lower in these than in similar segments obtained from control plants. The NaCl effect was salt-specific, and not osmotic, as incubation in 200 mM sorbitol did not reduce formazan staining intensity. SEZs elongation rates were higher in 200 mM sorbitol than in 100 mM NaCl, but the difference could be cancelled by scavenging or inhibiting O2*- production with 10 mM MgCl2 or 200 microM diphenylene iodonium, respectively. The actual ROS believed to stimulate growth is *OH, a product of O2*- metabolism in the apoplast. SEZ(s) elongation in 100 mM NaCl was stimulated by a *OH-generating medium. Fusicoccin, an ATPase stimulant, and acetate buffer pH 4, could also enhance elongation in these segments, although both failed to increase ROS activity. These results show that decreased ROS production contributes to the salinity-associated reduction in grass leaf elongation, acting through a mechanism not associated with pH changes.     

Does aluminium affect root growth of maize through interaction with the cell wall – plasma membrane – cytoskeleton continuum?

The mechanism of aluminium-induced inhibition of root elongation is still not well understood. It is a matter of debate whether the primary lesions of Al toxicity are apoplastic or symplastic. The present paper summarises experimental evidence which offers new avenues in the understanding of Al toxicity and resistance in maize. Application of Al for 1 h to individual 1 mm sections of the root apex only inhibited root elongation if applied to the first 3 apical mm. The most Al-sensitive apical root zone appeared to be the 1–2 mm segment. Aluminium-induced prominent alterations in both the microtubular (disintegration) and the actin cytoskeleton (altered polymerisation patterns) were found especially in the apical 1–2 mm zone using monoclonal antibodies. Since accumulation of Al in the root apoplast is dependent on the properties of the pectic matrix, we investigated whether Al uptake and toxicity could be modulated by changing the pectin content of the cell walls through pre-treatment of intact maize plants with 150 mM NaCl for 5 days. NaCl-adapted plants with higher pectin content accumulated more Al in their root apices and they were more Al-sensitive as indicated by more severe inhibition of root elongation and enhanced callose induction by Al. This special role of the pectic matrix of the cell walls in the modulation of Al toxicity is also indicated by a close positive correlation between pectin, Al, and Al-induced callose contents of 1 mm root segments along the 5 mm root apex. On the basis of the presented data we suggest that the rapid disorganisation of the cytoskeleton leading to root growth inhibition may be mediated by interaction of Al with the apoplastic side of the cell wall – plasma membrane – cytoskeleton continuum. This revised version was published online in June 2006 with corrections to the Cover Date.     

The reb1-1 mutation of Arabidopsis alters the morphology of trichoblasts,the expression of arabinogalactan-proteins and the organization of cortical microtubules

The root epidermal bulger 1 ( reb1) mutant of Arabidopsis thaliana (L.) Heynh. is characterized by a reduced elongation rate of the primary root and by the bulging of many, but not all, root epidermal cells. In this study, we investigated cell wall structure of root epidermal cells in reb1-1 by using serial sectioning, and light and electron microscopy in combination with immuno-cytochemistry and polysaccharide staining. We found that: (i) Cell bulging in the mutant was initiated in the zone of elongation of the root, and occurred exclusively in trichoblasts. (ii) reb1-1 and wild-type root cells stained identically with anti-pectin antibodies, such as JIM5. In contrast, the anti-arabinogalactan-protein antibodies, JIM14 and LM2, stained all epidermal cells in the wild type and trichoblasts preferentially, but in reb1-1 they stained the atrichoblasts only. (iii) Compared to the wild type, mutant trichoblasts had a thinner outer epidermal cell wall, which presented abnormal periodic acid-thio carbohydrazide silver proteinate (PATAg) staining. In addition, we investigated the organization of cortical microtubules in a reb1-1 mutant line expressing a green-fluorescent protein fused to a microtubule-binding domain from human microtubule-associated protein 4. Microtubules in the swollen trichoblasts of reb1-1 were either disordered or absent entirely. Together our findings indicate that the reb1-1 mutation results in an abnormal trichoblast cell wall, and suggest that cell surface arabinogalactan-proteins are required for anisotropic expansion and for orienting cortical microtubules.     

Disrupted actin dynamics trigger an increment in the reactive oxygen species levels in the Arabidopsis root under salt stress

Changes in actin dynamics represent the primary response of the plant cell to extracellular signaling. Recent studies have now revealed that actin remodeling is involved in abiotic stress tolerance in plants. In our current study, the relationship between the changes in actin dynamics and the reactive oxygen species (ROS) level at the initial stages of salt stress was investigated in the elongation zone of the Arabidopsis root tip. We found that a 200 mM NaCl treatment disrupted the dynamics of the actin filaments within 10 min and increased the ROS levels in the elongation zone cells of the Arabidopsis root tip. We further found that the NADPH oxidase activity inhibitor, diphenyleneiodonium, treatment blocked this ROS increase under salt stress conditions. The roles of actin dynamics and the NADPH oxidases in ROS generation were further analyzed using the actin-specific agents, latrunculin B (Lat-B) and jasplakinolide (Jasp), and mutants of Arabidopsis NADPH oxidase AtrbohC. Lat-B and Jasp promote actin depolymerization and polymerization, respectively, and both were found to enhance the ROS levels following NaCl treatment. However, this response was abolished in the atrbohC mutants. Our present results thus demonstrate that actin dynamics are involved in regulating the ROS level in Arabidopsis root under salt stress conditions. KEY MESSAGE: Salt stress disrupts the dynamics of the actin filaments in Arabidopsis in the short term which are involved in regulating the ROS levels that arise under salt stress conditions via the actions of the AtrbohC.     

Gibberellic acid and dwarfism effects on the growth dynamics of B73 maize (Zea mays L.) leaf blades: a transient increase in apoplastic peroxidase activity precedes cessation of cell elongation

The relationship between apoplastic peroxidase (EC 1.11.1.7) activity and cessation of growth in maize (Zea mays L.) leaf blades was investigated by altering elongation zone length. Apoplastic peroxidase activity in the elongation and secondary cell wall deposition zones of elongating leaf blades of the maize inbred line B73 was used as a control and compared to leaves of the dwarf mutant D8-81127, a near-isogenic line of B73 unresponsive to gibberellins, and to leaves of B73 plants to which gibberellic acid (GA(3)) had been applied via root uptake. Elongation zone length was increased by treatment with GA(3) through an increase in cell number as well as increased final cell length. The shorter elongation zone of dwarf leaves occurred primarily through reduced final cell length. Although elongation zone length differed among dwarf, control, and GA(3)-treated leaf blades, in all three treatments a transient increase in apoplastic peroxidase activity preceded a reduction in the segmental elongation rate in leaves. A peroxidase isoenzyme with pI 7.0 occurred in the leaf elongation zone during growth deceleration in all three treatments, and its activity decreased as growth displaced tissue into the region of secondary cell wall deposition. Growth cessation for all treatments coincided with the first appearance of peroxidase isozymes with pIs of 5.6 and 5.7. Based on the activity of particular isozymes relative to growth and differentiation, the pI 7.0 isoenzyme is most likely to be involved in cessation of cell elongation, while isozymes with pIs 5.6 and 5.7 are likely to be active in lignification.     

A mutant of Arabidopsis thaliana displaying altered patterns of cellulose deposition

A homozygous recessive mutant of Arabidopsis thaliana has been selected which displays altered patterns of cellulose deposition. The mutant was selected because leaf and stem trichomes lacked the strong birefringence under polarized light which is characteristic of plant cells which contain highly ordered cellulose in their secondary cell walls. Compared with wild-type A. thaliana, this mutant (designated tbr for trichome birefringence) also displays reduced birefringence in the xylem of the leaf. Direct chemical analyses of root, stem, and leaf tissues, including isolated leaf trichomes, support the conclusion that tbr is impaired in its ability to deposit secondary wall cellulose in specific cell types, most notably in trichomes where the secondary wall appears to be totally absent. Altered patterns of wound-induced callose deposition in trichomes and surrounding cells is another trait which also co-segregates with the tbr mutation.     

Differential Responsiveness of Cortical Microtubule Orientation to Suppression of Cell Expansion among the Developmental Zones of Arabidopsis thaliana Root Apex

Τhe bidirectional relationship between cortical microtubule orientation and cell wall structure has been extensively studied in elongating cells. Nevertheless, the possible interplay between microtubules and cell wall elements in meristematic cells still remains elusive. Herein, the impact of cellulose synthesis inhibition and suppressed cell elongation on cortical microtubule orientation was assessed throughout the developmental zones of Arabidopsis thaliana root apex by whole-mount tubulin immunolabeling and confocal microscopy. Apart from the wild-type, thanatos and pom2-4 mutants of Cellulose SynthaseA3 and Cellulose Synthase Interacting1, respectively, were studied. Pharmacological and mechanical approaches inhibiting cell expansion were also applied. Cortical microtubules of untreated wild-type roots were predominantly transverse in the meristematic, transition and elongation root zones. Cellulose-deficient mutants, chemical inhibition of cell expansion, or growth in soil resulted in microtubule reorientation in the elongation zone, wherein cell length was significantly decreased. Combinatorial genetic and chemical suppression of cell expansion extended microtubule reorientation to the transition zone. According to the results, transverse cortical microtubule orientation is established in the meristematic root zone, persisting upon inhibition of cell expansion. Microtubule reorientation in the elongation zone could be attributed to conditional suppression of cell elongation. The differential responsiveness of microtubule orientation to genetic and environmental cues is most likely associated with distinct biophysical traits of the cells among each developmental root zone.     

生长素类化合物及6－苯甲基腺嘌呤对拟南芥主根生长的抑制效应比较

为更好的研究生长素类化合物及6-苯甲基腺嘌呤（6－BA）对细胞分裂和细胞伸长的影响,以拟南芥主根为材料,从组织学水平比较了IAA、NAA、2,4－D和6－BA对拟南芥主根分生区和伸长区的抑制效应,发现IAA和NAA效果是相似的,可以通过促进细胞分裂显著增加根分生区长度,但也显著缩短主根仲长区长度,而2,4－D和6－BA则通过抑制细胞分裂来显著缩短根分生区长度,同时也显著缩短根伸长区的长度。     

Stunted plant 1 mediates effects of cytokinin, but not of auxin, on cell division and expansion in the root of Arabidopsis

Plants control organ growth rate by adjusting the rate and duration of cell division and expansion. Surprisingly, there have been few studies where both parameters have been measured in the same material, and thus we have little understanding of how division and expansion are regulated interdependently. We have investigated this regulation in the root meristem of the stunted plant 1 (stp1) mutation of Arabidopsis, the roots of which elongate more slowly than those of the wild type and fail to accelerate. We used a kinematic method to quantify the spatial distribution of the rate and extent of cell division and expansion, and we compared stp1 with wild type and with wild type treated with exogenous cytokinin (1 microM zeatin) or auxin (30 nM 2,4-dichlorophenoxyacetic acid). All treatments reduced average cell division rates, which reduced cell production by the meristem. Auxin lowered root elongation by narrowing the elongation zone and reducing the time spent by a cell in this zone, but did not decrease maximal strain rate. In addition, auxin increased the length of the meristem. In contrast, cytokinin reduced root elongation by lowering maximal strain rate, but did not change the time spent by a cell within the elongation zone; also, cytokinin blocked the increase in length and cell number of the meristem and elongation zone. The cytokinin-treated wild type phenocopied stp1 in nearly every detail, supporting the hypothesis that cytokinin affects root growth via STP1. The opposite effects of auxin and cytokinin suggest that the balance of these hormones may control the size of the meristem.     

Expression of genes for cell-wall proteins in dividing and wounded tissues ofZea mays L.

Hydroxyproline-rich glycoproteins (HRGPs) fromZea mays have been immunolocalized in the cell wall of root tip cells using ultrathin sections and antibodies ellicited against the purified protein. The accumulation of mRNA corresponding to this protein was studied using the cDNA probe. Maximum accumulation of the mRNA was found in tissues with a high proportion of dividing cells such as those in the root tip of young maize seedlings and a close relationship with cellular division was also observed in in-vitro cultures. However, the level of the mRNA in elongating tissues was minimal, as shown by studies carried out on the elongation zones of root tips and coleoptiles. The mRNA was induced by stress conditions, particularly by wounding young leaves and coleoptiles. It is concluded that in maize this group of proline-rich cell-wall proteins accumulates during cell division and not during cell elongation or differentiation, and participates in the stress-response mechanisms of the plant.     

盐胁迫条件下提高内源乙烯对拟南芥幼苗生长和根尖活性氧水平的影响

以模式植物拟南芥（Arabidopsis thaliana）为材料,研究了内源乙烯对幼苗耐盐性的影响。研究结果表明,在施加了浓度为100 mmol·L^-1的NaCl胁迫的基质环境中,野生型拟南芥幼苗的根长和根重都显著减小。在施加外源乙烯利后不仅能够缓解盐胁迫对幼苗根伸长生长的抑制作用,而且能够缓解盐胁迫对幼苗根增重生长的抑制作用。施加外源ACC则只能缓解盐胁迫对幼苗根增重生长的抑制作用,而不能缓解盐胁迫对根的伸长生长的抑制。此外,100 mmol·L^-1 NaCl的胁迫条件下,拟南芥幼苗根尖中ROS水平明显升高,而施加了乙烯利和ACC处理下,幼苗根尖ROS的水平在NaCl胁迫下并没有明显的升高,说明内源乙烯可以调控植物体内的ROS维持在正常的水平,使植物体免受氧化损伤,从而提高了幼苗耐盐性。