Quiescent center formation in maize roots is associated with an auxin-regulated oxidizing environment

Embedded within the meristem of all Angiosperm roots is a population of slowly dividing cells designated the quiescent center (QC). In maize roots the QC can constitute upwards of 800-1200 cells, most of which spend an extended period of time (180-200 hours) in the G(1) phase of the cell cycle. How the QC forms and is maintained is not known. Here we report that cells of the QC are characterized by their highly oxidized status. Glutathione and ascorbic acid occur predominately in the oxidized forms in the QC. This is contrasted with the status of these redox intermediates in adjacent, rapidly dividing cells in the root meristem, in which the reduced forms of these two species are favored. Using a redox sensitive fluorescent dye we were able to visualize an overall oxidizing environment in the QC, and we also made comparisons with the adjacent, rapidly dividing cells in the root meristem. Altering the distribution of auxin and the location of the auxin maximum in the root tip activates the QC, and cells leave G(1) and enter mitosis. Commencement of relatively more rapid cell division in the QC is preceded by changes in the overall redox status of the QC, which becomes less oxidizing. We discuss how the position of the auxin maximum may influence the redox status of the QC and thereby modulate the cell cycle.     

The quiescent center in roots of maize: initiation,maintenance and role in organization of the root apical meristem

Summary Using roots of maize, we tested the hypothesis that the origin and maintenance of the quiescent center (QC) are a consequence of polar auxin supply. Exposing roots to the polar auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA), or to low temperature (4 °C, with subsequent return to 24 °C), enhances mitotic frequency within the QC. In both treatments, the QC most typically is activated at its distal face, and the protoderm/dermatogen undergoes several periclinal divisions. As a result, the root body penetrates and ruptures the root cap junction and the characteristic closed apical organization changes to open. A QC persists during these changes in apical organization, but it is diminished in size. The data from the TIBA-treated roots suggest a role for auxin in the origin and maintenance of the QC, and further, that alterations in QC dimensions are a consequence of polar auxin supply. We hypothesize that the root cap, and specifically the root cap initials, are important in regulating polar auxin movements towards the root apex, and hence are important in determining the status of the QC.Abbreviations QC quiescent center - TIBA 2,3,5-triiodobenzoic acid Dedicated to the memory of Professor John G. Torrey     

Calcium Could Be Involved in Auxin-Regulated Maintenance of the Quiescent Center in the <Emphasis Type="Italic">Arabidopsis</Emphasis> Root

Ca²⁺ transduces hormone and environmental signals to the Ca²⁺ sensors and relays them to the downstream target effectors in cells. During the post-embryonic developmental process, auxin plays a critical role in maintaining the mitotically inactive status of the quiescent center (QC) and the root growth and development that follows. In this report, we demonstrate that Ca²⁺ plays an important role in the maintenance of the QC, probably by regulating PIN1-mediated auxin transport. Perturbation of the intracellular Ca²⁺ levels with chemicals that modify the Ca²⁺ level decreases the endogenous auxin level and the size of the auxin maximum in the root tip and, at the same time, activates QC cell division and expansion. This decreased level of auxin is almost completely restored to the control level by the treatment of exogenous auxin. Interestingly, treatment with Ca²⁺ level modifying chemicals significantly decreased the PIN1 expression in the root vasculature. Taken together, we suggest that balancing Ca²⁺ homeostasis is one of contributing factors in establishing the proper auxin maximum in the root tip and maintaining the QC identity.     

Auxin and ethylene interactions control mitotic activity of the quiescent centre, root cap size, and pattern of cap cell differentiation in maize

Root caps (RCs) are the terminal tissues of higher plant roots. In the present study the factors controlling RC size, shape and structure were examined. It was found that this control involves interactions between the RC and an adjacent population of slowly dividing cells, the quiescent centre, QC. Using the polar auxin transport inhibitor 1-N-naphthylphthalamic acid (NPA), the effects of QC activation on RC gene expression and border cell release was characterized. Ethylene was found to regulate RC size and cell differentiation, since its addition, or the inhibition of its synthesis, affected RC development. The stimulation of cell division in the QC following NPA treatment was reversed by ethylene, and quiescence was re-established. Moreover, inhibition of both ethylene synthesis and auxin polar transport triggered a new pattern of cell division in the root epidermis and led to the appearance of supernumerary epidermal cell files with cap-like characteristics. The data suggest that the QC ensures an ordered internal distribution of auxin, and thereby regulates not only the planes of growth and division in both the root apex proper and the RC meristem, but also regulates cell fate in the RC. Ethylene appears to regulate the auxin redistribution system that resides in the RC. Experiments with Arabidopsis roots also reveal that ethylene plays an important role in regulating the auxin sink, and consequently cell fate in the RC.     

Absence of a Causal Relationship between Auxin-Induced Growth and Changes in the Content of Ascorbic and Dehydroascorbic Acids in Excised Plant Tissues

The data reported indicate that the oxidation-reduction balance of the ascorbic acid system is not causally related to the auxin-regulation of cell elongation. There was no shift in the ascorbic acid (AA) to dehydroascorbic acid (DHA) ratio with growth-promoting concentration of auxin in several plant tissues. The AA to DHA ratio was experimentally increased without altering the growth rate. Inhibition of growth by supra-optimal auxin was associated with a decrease in the AA to DHA ratio. Since the AA to DHA ratio was lowered by EDTA treatment without altering growth, it seems unlikely that the decrease in the AA to DHA ratio related to the inhibition of growth by high levels of auxin.     

Role of Auxin-Induced Reactive Oxygen Species in Root Gravitropism

We report our studies on root gravitropism indicating that reactive oxygen species (ROS) may function as a downstream component in auxin-mediated signal transduction. A transient increase in the intracellular concentration of ROS in the convex endodermis resulted from either gravistimulation or unilateral application of auxin to vertical roots. Root bending was also brought about by unilateral application of ROS to vertical roots pretreated with the auxin transport inhibitor N-1-naphthylphthalamic acid. Furthermore, the scavenging of ROS by antioxidants (N-acetylcysteine, ascorbic acid, and Trolox) inhibited root gravitropism. These results indicate that the generation of ROS plays a role in root gravitropism.     

Localization of ascorbic acid, ascorbic acid oxidase, and glutathione in roots of Cucurbita maxima L

To understand the function of ascorbic acid (ASC) in root development, the distribution of ASC, ASC oxidase, and glutathione (GSH) were investigated in cells and tissues of the root apex of Cucubita maxima. ASC was regularly distributed in the cytosol of almost all root cells, with the exception of quiescent centre (QC) cells. ASC also occurred at the surface of the nuclear membrane and correspondingly in the nucleoli. No ASC could be observed in vacuoles. ASC oxidase was detected by immunolocalization mainly in cell walls and vacuoles. This enzyme was particularly abundant in the QC and in differentiating vascular tissues and was absent in lateral root primordia. Administration of the ASC precursor L-galactono-gamma-lactone markedly increased ASC content in all root cells, including the QC. Root treatment with the ASC oxidized product, dehydroascorbic acid (DHA), also increased ASC content, but caused ASC accumulation only in peripheral tissues, where DHA was apparently reduced at the expense of GSH. The different pattern of distribution of ASC in different tissues and cell compartments reflects its possible role in cell metabolism and root morphogenesis.     

Ethylene–auxin interactions regulate lateral root initiation and emergence in Arabidopsis thaliana

Plant root systems display considerable plasticity in response to endogenous and environmental signals. Auxin stimulates pericycle cells within elongating primary roots to enter de novo organogenesis, leading to the establishment of new lateral root meristems. Crosstalk between auxin and ethylene in root elongation has been demonstrated, but interactions between these hormones in root branching are not well characterized. We find that enhanced ethylene synthesis, resulting from the application of low concentrations of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), promotes the initiation of lateral root primordia. Treatment with higher doses of ACC strongly inhibits the ability of pericycle cells to initiate new lateral root primordia, but promotes the emergence of existing lateral root primordia: behaviour that is also seen in the eto1 mutation. These effects are correlated with decreased pericycle cell length and increased lateral root primordia cell width. When auxin is applied simultaneously with ACC, ACC is unable to prevent the auxin stimulation of lateral root formation in the root tissues formed prior to ACC exposure. However, in root tissues formed after transfer to ACC, in which elongation is reduced, auxin does not rescue the ethylene inhibition of primordia initiation, but instead increases it by several fold. Mutations that block auxin responses, slr1 and arf7 arf19, render initiation of lateral root primordia insensitive to the promoting effect of low ethylene levels, and mutations that inhibit ethylene-stimulated auxin biosynthesis, wei2 and wei7 , reduce the inhibitory effect of higher ethylene levels, consistent with ethylene regulating root branching through interactions with auxin.     

AUX1 promotes lateral root formation by facilitating indole-3-acetic acid distribution between sink and source tissues in the Arabidopsis seedling

Arabidopsis root architecture is regulated by shoot-derived signals such as nitrate and auxin. We report that mutations in the putative auxin influx carrier AUX1 modify root architecture as a result of the disruption in hormone transport between indole-3-acetic acid (IAA) source and sink tissues. Gas chromatography-selected reaction monitoring-mass spectrometry measurements revealed that the aux1 mutant exhibited altered IAA distribution in young leaf and root tissues, the major IAA source and sink organs, respectively, in the developing seedling. Expression studies using the auxin-inducible reporter IAA2::uidA revealed that AUX1 facilitates IAA loading into the leaf vascular transport system. AUX1 also facilitates IAA unloading in the primary root apex and developing lateral root primordium. Exogenous application of the synthetic auxin 1-naphthylacetic acid is able to rescue the aux1 lateral root phenotype, implying that root auxin levels are suboptimal for lateral root primordium initiation in the mutant.     

A role for mitochondria in the establishment and maintenance of the maize root quiescent center

Mitochondria in the oxidizing environment of the maize (Zea mays) root quiescent center (QC) are altered in function, but otherwise structurally normal. Compared to mitochondria in the adjacent, rapidly dividing cells of the proximal root tissues, mitochondria in the QC show marked reductions in the activities of tricarboxylic acid cycle enzymes. Pyruvate dehydrogenase activity was not detected in the QC. Use of several mitochondrial membrane potential (DeltaPsi(m)) sensing probes indicated a depolarization of the mitochondrial membrane in the QC, which suggests a reduction in the capacity of QC mitochondria to generate ATP and NADH. We postulate that modifications of mitochondrial function are central to the establishment and maintenance of the QC.     

Cell type identity in<Emphasis Type="Italic">Arabidopsis</Emphasis> roots is altered by both ascorbic acid-induced changes in the redox environment and the resultant endogenous auxin response

Redox plays a critical role in controlling many cellular processes of plant growth and development. To understand the effect of changes in redox on cell-type determination in the root meristem, we examined the influence of a strong reducing agent -ascorbic acid (AA) - on both the expression patterns of several cell type-specific promoters and the endogenous auxin sensitivity of auxin-responsive DR5::GUS transgenic plants. AA treatment altered the regular expression of columella-specific markers. Moreover, when the same treatment was applied to the DR5::GUS lines, normal expression of the GUS reporter was completely abolished in the auxin maximum, while exogenous auxin restored AA-driven depletion of that maximum. Interestingly, the level of DHA (dehydroascorbate, an oxidized form of AA) in the AA-treated roots was greatly increased. This indicates that changes in cell-type specificity and the sensitivity to endogenous auxin may result from an increase in the cellular DHA that is metabolized from exogenously supplied AA. Therefore, we propose that redox changes in the root meristem alter auxin homeostasis, perhaps causing a change in cell types within the root meristem.     

An Auxin Gradient and Maximum in the Arabidopsis Root Apex Shown by High-Resolution Cell-Specific Analysis of IAA Distribution and Synthesis

Local concentration gradients of the plant growth regulator auxin (indole-3-acetic acid [IAA]) are thought to instruct the positioning of organ primordia and stem cell niches and to direct cell division, expansion, and differentiation. High-resolution measurements of endogenous IAA concentrations in support of the gradient hypothesis are required to substantiate this hypothesis. Here, we introduce fluorescence-activated cell sorting of green fluorescent protein–marked cell types combined with highly sensitive mass spectrometry methods as a novel means for analyses of IAA distribution and metabolism at cellular resolution. Our results reveal the presence of IAA concentration gradients within the Arabidopsis thaliana root tip with a distinct maximum in the organizing quiescent center of the root apex. We also demonstrate that the root apex provides an important source of IAA and that cells of all types display a high synthesis capacity, suggesting a substantial contribution of local biosynthesis to auxin homeostasis in the root tip. Our results indicate that local biosynthesis and polar transport combine to produce auxin gradients and maxima in the root tip.