First published online January 27, 2006; 10.1104/pp.105.071977
Plant Physiology 140:1118-1125 (2006)
© 2006 American Society of Plant Biologists
DEVELOPMENT AND HORMONE ACTION
A Role for Mitochondria in the Establishment and Maintenance of the Maize Root Quiescent Center
Keni Jiang,
Tracy Ballinger,
Daisy Li,
Shibo Zhang and
Lewis Feldman*
Department of Plant and Microbial Biology, University of California, Berkeley, California 94720
 |
ABSTRACT
|
|---|
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 (  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.
Embedded in all angiosperm root apices is a population of slowly dividing cells that together form a region known as the quiescent center (QC). Depending on the species, the QC varies in size from four cells in Arabidopsis (Arabidopsis thaliana) to upward of 1,000 cells in the root apex of maize (Zea mays). On a biochemical level, one of the few known properties of the QC is its relatively oxidized redox status, which is reflected by the low concentrations in the QC of the reduced forms of glutathione and ascorbic acid, the two principal redox-regulating compounds in living systems (Jiang et al., 2003 ).
Cells comprising the QC spend a prolonged period in G1, dividing, on average, about once every 200 h (Clowes, 1961). Because divisions are infrequent and result in both a self-renewed QC cell and a sister cell that leaves the QC and repopulates the initial pool, a number of workers suggest that QC cells should be viewed as stem cells (Barlow, 1997; Ivanov, 2004; Jiang and Feldman, 2005). How this balance between self-renewing divisions and differentiation is regulated is not understood. However, recent work with animal stem cells points to redox state as having a central role in modulating this equilibrium (Smith et al., 2000).
We have hypothesized that the QC is established and maintained as a consequence of auxin that is polarly transported to the root tip, where it can accumulate to relatively high levels leading to the oxidized state in the QC (Jiang and Feldman, 2003, 2005). How auxin influences redox status is not known. But what is clear is that oxidative stress can compromise many cellular activities, including, as has been reported, mitochondrial function (Jiménez et al., 1998). Depending on the severity of the oxidized stress, mitochondria can respond in different ways, including a reduction in the flux capacities of the tricarboxylic acid (TCA) cycle, thereby affecting the synthesis of ATP and NADH/NADPH and, as a consequence, affecting energy-dependent activities, including cell division. Stress-induced reductions in mitochondrial function have been visualized through the use of dyes whose extent of accumulation in mitochondria reflects mitochondrial activity (Smith et al., 2000).
Thus, given the known oxidized redox status of the QC and reports of compromised mitochondrial functioning under conditions of oxidative stress, we investigated whether alterations in mitochondrial activity occur in the QC. Here we report differences between mitochondria in the slowly dividing QC cells compared to mitochondria in the adjacent, rapidly dividing cells of the proximal meristem (PM). We suggest that these changes in mitochondrial function may underlie the establishment and maintenance of the QC and, as well, link auxin and oxidative stress.
|
RESULTS
|
|---|
Three approaches were taken to characterize mitochondria in the QC: (1) examining mitochondrial ultrastructure; (2) evaluating mitochondrial physiological activity; and (3) evaluating the levels, status, and processing of specific mitochondrial transcripts (either nuclear or mitochondrial encoded).
QC Mitochondria Show Normal Structure
Mitochondria in the QC were characterized and compared to like organelles in the adjacent, rapidly dividing cells bordering on the proximal face of the QC (the PM; Jiang and Feldman, 2005; Fig. 1
, A and B). QC mitochondria are egg-to-oval shaped, numerous, and encircle the nucleus (Fig. 1, C and D). A continuously intact, double membrane is evident, as are cristae (Fig. 1D). As in the QC, mitochondria in the PM encircle the nucleus (Fig. 1E). However, in contrast to QC mitochondria, PM mitochondria frequently assumed a dumbbell shape (in approximately two-thirds [eight] of the roots examined ultrastructurally), and this is interpreted as indicative of their capacity to divide (Fig. 1, E and F). A delimiting double membrane is evident, as are cristae that extend inward, about one-third to one-half of the diameter of the mitochondrion (Fig. 1F, arrow).
View larger version (173K):
[in this window]
[in a new window]
|
Figure 1. Longitudinal sections of the maize root tip. A, Light micrograph indicating the locations of the QC, PM, and root cap (RC). B to F, Transmission electron micrographs of the maize root tip. B, Longitudinal section of the maize root tip/QC at low magnification. The RC was removed just prior to fixation. QC cells marked with an asterisk (*) were examined further at higher magnification (C and D). Note cell files converging in the QC. C, QC cell in longitudinal section. Note large number of round-to-oval-shaped mitochondria (arrow) encircling the nucleus (N). D, High-power view of C showing that QC mitochondria (arrow) have intact double membranes and cristae. E and F, Cells from the PM. Note the relatively thin walls (indicative of a relatively high rate of mitosis), and dumbbell-shaped mitochondria (arrow) clustered about the nuclei.
|
|
Reconfirmation of the Oxidized Status of the QC
We reconfirmed the relatively oxidized redox status of the QC through the use of H2DCFDA, a dye that is oxidized in the presence of peroxides (Collins et al., 2000) and that we previously used to assess QC redox status (Jiang et al., 2003). QCs are treated with the reduced form of H2DCFDA, which when supplied is colorless. As the dye is oxidized, it fluoresces in UV light and thereby points to a relatively oxidizing microenvironment in the QC (Fig. 2A
).
View larger version (148K):
[in this window]
[in a new window]
|
Figure 2. Whole mounts of QCs treated with various probes. Scale bar = 100 µm (except in C where the scale bar = 25 µm). A, Treated with carboxy-H2DCFDA (C-400). Bright staining in the center of the QC points to the relatively oxidized redox status of this region. B and C, Whole mounts of a QC treated with the mitochondrial marker dye, Mitotracker Orange. Fluorescing mitochondria occur at the margins, but not in the center, of the QC. C, High-magnification view of B showing a strip of cells extending from the outside edge of the QC to the center (*). Fluorescing mitochondria are evident clustered around nuclei (arrow). D and E, Whole mounts of two different QCs treated with the dye JC-9. Fluorescing mitochondria (numerous small white dots) occur in cells at the margins, but not in the center (*), of the QC. Bright, diffuse staining is artifact.
|
|
Mitochondrial Membrane Potential Differs in the QC
JC-9 and MitoTracker Orange are two mitochondrial membrane potential (m) sensing probes that are used to indicate the cellular energy levels of mitochondria (Duchen et al., 1993; Sureau et al., 1993; Cossarizza et al., 1994; Castedo et al., 1996). Both probes accumulate in mitochondria that maintain a membrane potential. MitoTracker Orange is particularly useful because once it enters the mitochondrion it reacts with thiols on proteins and peptides to form an aldehyde-fixable conjugate. An absence or low fluorescence of the dye in the mitochondrion indicates a depolarization of the inner mitochondrial membrane and thereby suggests a reduction in the capacity of these mitochondria to generate ATP (Waterhouse et al., 2001). When QCs are treated with these probes, mitochondria in cells at the margins of the QC accumulate the dye and fluoresce, whereas mitochondria in central QC cells show no fluorescence (Fig. 2, B–E). The pattern of mitochondrial staining is nearly identical for both probes and thereby points to an absence or marked reduction of mitochondrial membrane potential (m) in central QC cells and suggests, therefore, a decrease in the capacity of these cells to generate energy-rich reductants.
TCA Cycle Enzyme Activities and Substrates Are Lowered in the QC
We assayed the activities of a number of TCA cycle enzymes, including pyruvate dehydrogenase (PDH),  -ketoglutarate dehydrogenase (KDH), succinate dehydrogenase (SDH), malate dehydrogenase (MDH), and malic enzyme (ME). Except for PDH, activities for all other enzymes are detected in both QC and PM extracts. When normalized to total protein per milligram of tissue, individual enzymes were 3 to 10 times more active in the PM compared to the QC (Table I
). Notable is the absence of any measurable PDH activity in the QC. Pyruvate concentrations also differed between the two tissues; pyruvate was approximately 8 to 10 times more concentrated in the PM (12 ± 2 mM mg–1) compared to the QC (1.5 ± 0.7 mM mg–1).
View this table:
[in this window]
[in a new window]
|
Table I. Comparison of TCA enzyme activities in the QC and PM
Activities are measured spectrophotometrically (see "Materials and Methods" for specifics on each enzyme) and normalized to 100 µg of total protein. Values are means ± SD (n = 3). For each enzyme, mean values of the QC and PM were compared using Student's t test and found to be significant at the 95% confidence level (P < 0.05).
|
|
Expression of Nuclear-Encoded Transcripts for Mitochondrial Proteins Is Reduced in the QC
Based on the observation that nuclear-encoded transcripts of mitochondrial proteins may be altered by stress (Giegé et al., 2005), we investigated whether the reduced activities in the QC of TCA cycle enzymes (Table I) could be related to reductions in transcription. We measured the levels of transcripts in the QC for three nuclear-encoded TCA cycle-related proteins (or protein subunits; PDH, SDH, and ME) and compared their levels to those in the adjacent PM. We found that all three transcripts show a 20% to 30% reduction in expression in the QC (relative to the total mRNA) compared to their levels in the adjacent PM (Table II
).
QC and PM Cells Have a Similar Editing Status for Mitochondrial mRNAs
Mitochondrial transcripts (mtRNA) are typically extensively edited, which involves the conversion of cytidines to uridines (Gagliardi and Gualberto, 2004). We investigated the status of mtRNA editing in the QC by examining specific mitochondrial transcripts for which editing has already been reported in maize, namely, ribosomal protein S13 (RSP13; GenBank accession no. AF079549; Williams et al., 1998) and ATP synthase subunit 9 (ATP9; GenBank accession no. AF390542; Grosskopf and Mulligan, 1996). In both the QC and PM, ATP9 is completely edited, as has previously been reported for this transcript in other maize tissues (suspension-cultured cells and seedlings; Grosskopf and Mulligan, 1996). We sequenced five QC cDNA ATP9 clones; all showed complete editing at the seven editing sites characterized in maize by Grosskopf and Mulligan (1996). In contrast to ATP9, RSP13 was incompletely edited (Table III
). This gene has six C-to-U editing sites, of which Williams et al. (1998) report that 70% of the examined 30 cDNA clones were edited in all six sites, and 3% were completely unedited. We sequenced six RSP13 cDNA clones from both the QC and PM (a total of 12). Two of the six (33%), either from the PM or the QC, were incompletely edited (none showed no editing), whereas four of the six (66%) were completely edited in both the QC and PM. Thus, we conclude that the mtRNA editing process is similar in both the QC and PM and is consistent with other reports of the editing status of these mitochondrial transcripts in maize (Grosskopf and Mulligan, 1996; Williams et al., 1998).
View this table:
[in this window]
[in a new window]
|
Table III. Editing status of ribosomal protein 13 (RSP13) mitochondrial transcripts in the QC and PM
+, C-to-T change at the editing site; –, no editing at that position in the cDNA.
|
|
|
DISCUSSION
|
|---|
The maize QC is composed of cells that spend an extended period of time (>180 h) in G1. The reasons for this prolonged G1 are not known, but because these cells are under oxidative stress (Jiang et al., 2003; Fig. 2A), we considered the possibility that reactive oxygen species may mediate the induction of G1 cell cycle arrest, as has been reported for other systems (Hutter et al., 1997; Reichheld et al., 1999; Smith et al., 2000; Boonstra and Post, 2004). In mice, sublethal doses of hydrogen peroxide induce a rapid, transient growth arrest in fibroblasts by activating cell cycle checkpoints at G1 and S (Barnouin et al., 2002), and in human colonic CaCo-2 cells, Noda et al. (2001) report that mild intracellular redox imbalance can inhibit the G1-S transition. Toward elucidating the mechanism of G1 arrest in the QC, we considered reports noting that mitochondria are very sensitive to oxidative stress (Fujie et al., 1993), which can cause damage to mitochondrial components (Valls et al., 1994; Beyer et al., 1996; Brady et al., 2004), leading to a disruption of mitochondrial membrane potential (m), an attendant impairment in mitochondrial function, and cell cycle arrest (Yoon et al., 2005). Based on this scenario, we investigated whether G1 cell cycle arrest in the QC is associated with an alteration in mitochondrial function, including alterations in membrane potential (m), mitochondrial physiology, and perhaps even structure.
An Absence of Membrane Potential Suggests That QC Mitochondria Are Compromised in Their Capacity to Generate ATP
The absence of uptake and/or oxidation of mitochondrial membrane marker dyes (JC-9 and MitoTracker Orange) into the central region of the QC (Fig. 2, B–E) points to a reduction in mitochondrial membrane potential (m; Duchen et al., 1993; Sureau et al., 1993; Cossarizza et al., 1994; Castedo et al., 1996) and thereby suggests a reduced capacity of these mitochondria to generate ATP and NADH (Duchen, 2004). These conclusions are supported by earlier work reporting a lower energy status of the QC compared to cells in the adjacent PM (Jiang et al., 2003). Because dividing cells generally must maintain a minimal ATP content to satisfy the G1-S checkpoint energy requirement (Sweet and Singh, 1995), redox regulation of ATP production in the mitochondria may be central to the maintenance of the QC; less ATP/NADH could underlie the decrease in cell division in the QC.
An Oxidized Redox Status Correlates with Lower TCA Enzyme Activities in the QC
Our data (Table I) showing reductions in the activities of TCA cycle enzymes in QC mitochondria compared to mitochondria in more rapidly dividing, adjacent root meristem cells (PM) support the view that an oxidized environment can correlate with changes in activities of mitochondrial proteins (Sweetlove et al., 2002). In Arabidopsis, Sweetlove et al. (2002) have investigated the impact of oxidative stress on mitochondria and have shown that, under oxidizing conditions, proteins associated with the TCA cycle were less abundant and that oxygen consumption was significantly decreased, suggesting less ATP/NADH production. Based on the key role that PDH plays in providing the entry substrate (acetyl-CoA) for the TCA cycle, we suggest that the decrease in PDH activity (none detected in the QC; Table I) may be central to an overall reduction in TCA cycle activities in the QC. Exactly how PDH activity might be reduced is not known. However, the complexity and multiple subunits of PDH and its specific cofactor requirements make it a possible target in the QC for oxidative inactivation, as has already been demonstrated for PDH in other systems (Cabiscol et al., 2000; Holness and Sugden, 2003; Garg et al., 2004; Martin et al., 2005). Moreover, the significant reduction in available pyruvate in the QC (8–10 times less in QC compared to PM) strengthens our contention that alterations in PDH activity could account for a diminution in mitochondrial functioning in the QC. As well, these differences in enzyme activities between mitochondria in the QC and PM support the notion that mitochondria can be primary intracellular targets for the initiation of changes in cell function (Fujie et al., 1993; Smith et al., 2000).
QC Mitochondria Are Undamaged and Have Normal Structure
Oxidative stress and a reduction in mitochondrial membrane potential (m) are often associated with apoptosis (Mancini et al., 1997). However, in the QC, an oxidizing environment does not lead to cell death. Rather, the overall cellular ultrastructure of the QC, including that of the mitochondria, is fairly typical of that found in a variety of unstressed plant cells (Fig. 1, B–D). Cristae are present, but small, as has been previously reported for other maize root cells (Clowes and Juniper, 1964). The intactness of the mitochondria in the QC contrasts with what is observed in like organelles in tissues undergoing senescence in which there is a loss of mitochondrial membrane integrity and organization (Pastori and del Río, 1994). Thus, the observation that mitochondria number and ultrastructure in the QC are normal and unchanged from that in adjacent, dividing cells implies that these redox-sensitive organelles are not undergoing apoptosis/senescence in the QC. This also implies that, in the QC, as has been reported for other plant systems (Millar et al., 2001; Møller, 2001; Dutilleul et al., 2003), there must be some mechanism for ameliorating the effects of oxidative stress on these characteristically reactive oxygen species-sensitive organelles. In this regard, the fact that reduced glutathione and ascorbate are still present in the QC (Jiang et al., 2003) suggests a possible mechanism for protecting against severe oxidative stress and thus may provide insight about why QC mitochondria are apparently undamaged and do not undergo apoptosis, namely, that reduced forms of redox regulators (e.g. glutathione, ascorbate) are never absent or are maintained in a ratio with their oxidized forms so as to preclude apoptosis. Thus, because oxidative stress, depending on its severity, can lead to different developmental pathways (Arimura et al., 2003) including apoptosis, this suggests that the QC may avoid the apoptotic pathway by actively modulating the levels of oxidative stress, as recently suggested for other plant tissues (Dutilleul et al., 2003). The specific mechanisms underlying this tolerance are not completely known but likely involve up-regulation of the antioxidant system at the transcript level (Dutilleul et al., 2003). And because many of these genes are nuclear encoded, this implies cross talk between the mitochondria and other organelles. In this way, redox acclimation can extend far beyond the mitochondria.
Expression of Nuclear-Encoded Mitochondrial Transcripts Is Down-Regulated in the QC
The levels of nuclear-encoded transcripts for TCA cycle-related proteins (PDH, SDH, and ME) are reduced in the oxidized microenvironment of the QC (Table II), paralleling the reduction in measurable activity in the QC of the respective proteins (Table I). This suggests that a reduction in levels of nuclear-encoded mitochondrial transcripts could underlie the changed physiology of mitochondria in the QC, including a reduction in mitochondrial membrane potential (m). Giegé et al. (2005) advanced similar ideas to account for the decrease in mitochondrial biogenesis and function in Suc-starved Arabidopsis cell cultures. In this system, nutrient deprivation resulted in a decrease in transcripts for nuclear-encoded mitochondrial proteins, leading to the suggestion that the availability of nuclear-encoded subunits could be a limiting factor in mitochondrial function. Of particular relevance in that work is the down-regulation of genes for pyruvate metabolism, including PDH and ME, leading Giegé et al. (2005) to suggest that, in Suc-starved cells, "the abundance of nuclear transcripts could become the limiting factor in the synthesis and assembly of functional mitochondrial complexes" (p. 1507). Although we have no evidence that reduced mitochondrial transcript abundance leads to impairment in mitochondrial function in the QC, we, however, can conclude that a reduction in mitochondrial transcript levels does not result in any changes to organelle structure, which is different from what happens to mitochondria in Suc-stressed cells (Giegé et al., 2005).
Mitochondrial Transcripts in the QC Are Edited Normally
RNA editing of mitochondrial transcripts is required to produce a functional gene product (Lupold et al., 1999; Giegé et al., 2000; Nakajima and Mulligan, 2001). Thus because aberrant, incompletely edited transcripts can disrupt the function of the organelle gene expression system and compromise the metabolic processes of mitochondria, we investigated the status of RNA editing in QC mitochondria. From examining several already well-characterized maize mitochondrial transcripts (Grosskopf and Mulligan, 1996; Williams et al., 1998), we conclude that any differences between mitochondria in the QC and PM are not due to differences in editing, which is the same in both tissues. This again supports our view that mitochondria in the QC are undamaged and are not undergoing apoptosis. Moreover, the similarities between our data for RSP13 of edited versus not edited (66% versus 33%) agree well with that reported earlier by Williams et al. (1998) for maize, and thus again emphasize the point that mitochondria in the QC are not altered in their processing of mRNA.
Reduced Mitochondrial Activity May Be Related to the Stem Cell Nature of the QC
Because of their apparent ability for unlimited proliferation, self maintenance, and self renewal, a number of workers conclude that QC cells should be viewed as stem cells (Barlow, 1997; Ivanov, 2004; Jiang and Feldman, 2005). Here we raise the possibility that at least some of this stemness may be related to reduced mitochondrial activity. Reports that intracellular redox state can influence the balance between self renewal and differentiation in a variety of stem cell/precursor cell populations (Smith et al., 2000; Noda et al., 2001; Barros et al., 2004) imply a role for mitochondria in determining the state of cell differentiation. Using the dye rhodamine 123, which is reflective of mitochondrial activity, Smith et al. (2000) showed a lesser extent of mitochondrial labeling in hematopoietic stem cells and in hepatic precursor cells. These workers thus suggested that "redox modulation functions in multiple processes related to self renewal and differentiation" (Smith et al., 2000; p. 10037). As well, work showing that mild mitochondrial uncoupling is able to increase both the chronological and replicative life span of cells (Barros et al., 2004) supports the contention that redox affects the state/level of cell differentiation via a mechanism involving mitochondria. It is intriguing to speculate here that the stem cell nature of QC cells could also be related to their reduced mitochondrial activity.
|
CONCLUSION
|
|---|
Previously we provided evidence that auxin imposes an oxidative stress on the root tip and we hypothesized that the formation of a QC is an inevitable developmental outcome of this oxidized microenvironment (Jiang et al., 2003; Jiang and Feldman, 2005). Here we elaborate on that scenario and suggest that altered mitochondrial membrane potential and changed TCA cycle biochemistry reflect responses of mitochondria to oxidative stress and result in the establishment of a redox homeostasis that underlies the formation and maintenance of the QC.
|
MATERIALS AND METHODS
|
|---|
Plant Growth Conditions and Tissue Collection
Maize caryopses (Zea mays var. Merit; Asgrow Seed) were imbibed and germinated in the dark at 25°C for 2 to 3 d. For experiments to measure TCA cycle enzymes and cycle intermediates, QCs, and the adjacent zone of rapidly dividing cells, the PM, were collected separately (as described in Feldman and Torrey, 1976) and assayed as described below. In this cultivar, the QC is separable from the PM as a consequence of a weak, thin-walled junction.
Extraction and Assay of Pyruvate and TCA Enzymes
The activities of five TCA cycle-associated enzymes were measured: KDH (Nulton-Persson and Szweda, 2001), PDH (Randall, 1982), NAD+ ME (Geer et al., 1980), MDH (Nunes-Nesi et al., 2005), and SDH (Strain et al., 1998). For each assay, 60 to 70 QCs (approximately 0.42 mg) and 20 PMs (approximately 11 mg) were homogenized in 70 or 150 µL, respectively, of the appropriate extraction buffer. The final volume for each assay was 75 to 150 µL. For all assays, more or less equal amounts of total protein were used and the activities expressed on a basis of 100 µg total protein. All assays were replicated at least three times and the results expressed as a mean ± SD. For analysis limited to two groups (QC and PM), Student's t test was employed (P < 0.05). Spectrophotometric measurements were made on a Shimadzu UV160U spectrophotometer using Eppendorf Uvette microcuvettes. The total amount of pyruvate was determined as described by Millenaar et al. (1998).
Assaying Oxidative Activity (Oxidative Stress) in Living Tissues
Assaying oxidative activity in living cells was assessed using a dye that is colorless when chemically reduced (when freshly obtained) but, when oxidized, fluoresces in UV light (340-nm irradiation; 530-nm emission). For this work, we used carboxy-H2DCFDA (C-400) dye (Molecular Probes; catalog no. C–6827) as previously described (Jiang et al., 2003).
Assaying Mitochondrial Membrane Potential (Mitochondrial Activity)
To ascertain the functional status of mitochondria in the QC, we used two mitochondria-selective dyes, JC-9 (3,3'-dimethyl-b-naphthoxazolium iodide; Invitrogen; catalog no. D22421) and MitoTracker Orange (Invitrogen; catalog no. M7510). Uptake of these dyes occurs in functioning mitochondria and is dependent on the establishment and maintenance of mitochondrial membrane potential (m; Duchen et al., 1993; Sureau et al., 1993; Cossarizza et al., 1994; Castedo et al., 1996). Because of the cell wall, entry of these dyes into the plant protoplasts is usually retarded. Thus, to increase dye uptake, QCs were first excised and placed on a glass slide in a drop of solution containing protoplasting enzymes (3% cellulase, 1%–3% pectinase; Onazuka) in 10 mM potassium-phosphate buffer, pH 5.7. Treatments were brief, usually 15 to 30 min. Following this limited enzyme exposure, the QCs were washed several times with plain buffer and then incubated for 2 h in 0.012 mM JC-9 dissolved in 10 mM potassium-phosphate buffer, pH 5.7, or for 1 to 2 h in 0.1 to 0.25 nM Mitotracker Orange dissolved in 10 mM potassium-phosphate buffer, pH 5.7. Following incubation with the dyes, the QCs were washed several times in plain buffer and then observed under UV light using a Leica DM microscope.
Electron Microscopy
All solutions were made with 0.05 M sodium cacodylate buffer, pH 7.2. Just before fixation, root caps were excised from the root tips so as to permit more rapid entrance of the fixative into the QC. Immediately after decapping, root tips (1 mm) were fixed at room temperature for 6 h in 6% glutaraldehyde, rinsed three times for 15 min in buffer, followed by postfixation and staining with 1% osmium tetroxide for 1 to 2 h. Following three 5-min rinses with buffer and distilled water (three 10-min rinses), the tissues were dehydrated through a graded ethanol series. After dehydration, the tissues were embedded in Spurr's resin. Approximately 60-nm-thick longitudinal sections were cut using a RMC MT6000 microtome placed on slot grids and stained for 10 min in 2% uranyl acetate followed by a 5-min staining in either Reynold's lead citrate or Sato's lead. The stained sections were viewed using a FEI Tecnai 12 120-kV transmission electron microscope. A total of 12 roots tips, representing three separate fixations, were examined ultrastructurally.
RNA Editing
RNA Isolation
For each experiment, pooled tissues (approximately 180–200 PMs or 1,400–1,500 QCs) were homogenized in liquid nitrogen and total RNA extracted using the RNAwiz kit (Ambion) according to the manufacturer's protocol.
Dnase I-Treated RNA
Total RNA (15–17 µg) was digested in a 100-µL reaction volume with 10 units of RNAse-free DNase I (Gibco BRL) for 1 h at 37°C, followed by two extractions: the first with 100 µL of phenol:chloroform:isoamyl alcohol (25:24:1, v/v) and the second with 100 µL of chloroform:isoamyl alcohol (24:1, v/v). The RNA was then precipitated by adding 10 µL of 3 M NaOAc plus 275 µL of ethanol (100%), and incubated for 30 min at –80°C followed by centrifuging for 30 min at 10,000g. The resulting pellet was washed with 200 µL of 80% ethanol and resuspended in 20 µL of diethylpyrocarbonate-treated water. RNA integrity was assessed by examining the rRNA band after electrophoresis on a 1% agarose gel.
Reverse Transcription-PCR and Sequencing to Determine the Editing Status of mtRNA
For detecting the editing status of the mtRNA, 1 µg of total RNA from either PM or QC tissue was reverse transcribed using SuperScript RNase H– reverse transcriptase (Invitrogen) with a gene-specific 3' antisense oligonucleotide in a 10-µL reaction volume. For RSP13, we used the oligonucleotide designated MMM2 (Williams et al., 1998) and for ATP9 we used the oligonucleotide designated ZmATP9-3' (Grosskopf and Mulligan, 1996) as previously described (Williams et al., 1998). For each sample, a negative control was carried out in which the reverse transcriptase was omitted, allowing for assessment of genomic DNA contamination. Then, the reactions were diluted four times and 1 µL of this product was amplified by PCR using gene-specific primers for RSP13 (MMM1 and MMM2; Williams et al., 1998) and for ATP9 (ZmATP9-3' and ZmATP9-5'; Grosskopf and Mulligan, 1996). Amplification was performed for 25 cycles of 20 s at 94°C, 30 s at 60°C, and 45 s at 72°C. PCR products were isolated from the gel and cloned in the pGEM vector (Promega), which was used to transform Escherichia coli. Following transformation, six colonies from either the QC or PM were randomly selected and the plasmids were isolated and sequenced.
Measuring the Levels of Selected Nuclear-Encoded Mitochondrial Transcripts Using Real-Time Quantitative Reverse Transcription-PCR
Total RNA was quantified spectrophotometrically. As an internal standard for normalization, we added approximately 150 pg of kanamycin 1.2-kb control RNA (Kan 1.2; Promega) per 30 µg of total RNA in a total volume of 60 µL to give a final Kan 1.2 concentration of 2.5 pg µL–1, as described by McMaugh and Lyon (2003). The first strand of cDNA was synthesized from 2 µL of the Kan 1.2-spiked total RNA using an oligo dT(18) primer and SuperScript II RNase H– reverse transcriptase (Invitrogen). For each sample, an additional control reaction was carried out in which the reverse transcriptase was omitted, thereby allowing for assessment of genomic DNA contamination. The cDNA solutions and the controls were diluted to 40 µL with DNase-free water before PCR.
Quantitative PCR was performed as previously described (Jiang et al., 2006). In each experiment, two standard curves, one for the target gene and one for the Kan 1.2, were prepared from root-tip tissue using a cDNA dilution series (1x, 0.5x, 0.3x, 0.25x, and 0.15x). All samples were assayed in triplicate. For each standard curve, the linearity (Pearson correlation coefficient) was higher than 0.900. Target cDNAs were normalized to Kan 1.2. Differences in transcript levels between the QC and PM are expressed as a ratio (QC:PM; Table II). The results are presented as the means of two independent PCR experiments. A positive control was run on AUX1 (GenBank accession no. AJ011794), which is up-regulated in the QC (Hochholdinger et al., 2000), and a negative control was run on GAPC4, which is down-regulated in the QC, as determined by virtual northern blots (data not shown). Virtual northern blots (SMART kit; CLONTECH) were performed as previously described (Jiang et al., 2006). All gene-specific primers are indicated in Table IV
, except for GAPC4 and Kan 1.2, which have both been previously described by Jiang et al. (2006) and McMaugh and Lyon (2003), respectively.
Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AF069911, AF079549, AF390542, AJ011794, AJ012374, and AW037138.
|
ACKNOWLEDGMENTS
|
|---|
We are grateful for the assistance of the staff of the University of California, Berkeley, Electron Microscopy Facility, with special thanks to Reena Zalpuri.
Received September 27, 2005;
returned for revision January 15, 2006;
accepted January 17, 2006.
|
FOOTNOTES
|
|---|
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Lewis Feldman (feldman{at}nature.berkeley.edu).
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.071977.
* Corresponding author; e-mail feldman{at}nature.berkeley.edu; fax 510–642–4995.
|
LITERATURE CITED
|
|---|
Arimura T, Kojima-Yuasa A, Watanabe S, Suzuki M, Kennedy DO, Matsui-Yuasa I (2003) Role of intracellular reactive oxygen species and mitochondrial dysfunction in evening primrose extract-induced apoptosis in Ehrlich ascites tumor cells. Chem Biol Interact 145: 337–347[CrossRef][ISI][Medline]Barlow PW (1997) Stem cells and founder zones in plants, particularly their roots. In CS Potten, ed, Stem Cells. Academic Press, London, pp 29–57 Barnouin K, Dubuisson ML, Child ES, Fernandez de Mattos S, Glassford J, Medema RH, Mann DJ, Lam EW (2002) H2O2 induces a transient multi-phase cell cycle arrest in mouse fibroblasts through modulating cyclin D and p21Cip1 expression. J Biol Chem 277: 13761–13770[Abstract/Free Full Text] Barros MH, Bandy B, Tahara EB, Kowaltowski AJ (2004) Higher respiratory activity decreases mitochondrial reactive oxygen release and increases life span in Saccharomyces cerevisiae. J Biol Chem 279: 49883–49888[Abstract/Free Full Text] Beyer RE, Segura-Aguilar J, Di Bernardo S, Cavazzoni M, Romana F, Fiorentini D, Galli MC, Setti M, Landi L, Lenaz G (1996) The role of DT-diaphorase in the maintenance of the reduced antioxidant form of coenzyme Q in membrane systems. Proc Natl Acad Sci USA 93: 2528–2532[Abstract/Free Full Text] Boonstra J, Post JA (2004) Molecular events associated with reactive oxygen species and cell cycle progression in mammalian cells. Gene 337: 1–13[CrossRef][ISI][Medline] Brady NR, Elmore SP, van Beek JJHGM, Krab K, Courtoy PJ, Hue L, Westerhoff HV (2004) Coordinated behavior of mitochondria in both space and time: a reactive oxygen species-activated wave of mitochondrial depolarization. Biophys J 87: 2022–2034[Abstract/Free Full Text] Cabiscol E, Piulats E, Echave P, Herrero E, Ros J (2000) Oxidative stress promotes specific protein damage in Saccharomyces cerevisiae. J Biol Chem 35: 27393–27398 Castedo M, Hirsch T, Susin SA, Zamzami N, Marchetti P, Macho A, Kroemer G (1996) Sequential acquisition of mitochondrial and plasma membrane alterations during early lymphocyte apoptosis. J Immunol 157: 512–521[Abstract] Clowes FAL (1961) Apical Meristems. Blackwell Scientific Publications, Oxford Clowes FAL, Juniper BE (1964) The fine structure of the quiescent centre and neighbouring tissues in root meristems. J Exp Bot 15: 622–630[Abstract/Free Full Text] Collins TJ, Lipp P, Berridge MJ, Li W, Bootman MD (2000) Inositol 1,4,5-trisphosphate-induced Ca2+ release is inhibited by mitochondrial depolarization. Biochem J 347: 593–600[CrossRef][ISI][Medline] Cossarizza A, Kalashnikova G, Grassilli E, Chiappelli F, Salvioli S, Capri M, Barbieri D, Troiano L, Monti D, Franceschi C (1994) Mitochondrial modifications during rat thymocyte apoptosis: a study at the single cell level. Exp Cell Res 214: 323–330[CrossRef][ISI][Medline] Duchen MR (2004) Roles of mitochondria in health and disease. Diabetes 53: S96–S102[Abstract/Free Full Text] Duchen MR, McGuinness O, Brown LA, Crompton M (1993) On the involvement of a cyclosporin A sensitive mitochondrial pore in myocardial reperfusion injury. Cardiovasc Res 27: 1790–1794[Free Full Text] Dutilleul C, Garmier M, Noctor G, Mathieu C, Chétrit P, Foyer CH, de Paepe R (2003) Leaf mitochondria modulate whole cell redox homeostasis, set antioxidant capacity, and determine stress resistance through altered signaling and diurnal regulation. Plant Cell 15: 1212–1226[Abstract/Free Full Text] Feldman LJ, Torrey JG (1976) The isolation and culture in vitro of the quiescent center of Zea mays. Am J Bot 63: 345–355[CrossRef][ISI] Fujie M, Kuroiwa H, Suzuki T, Kwano S, Kuroiwa T (1993) Organelle DNA synthesis in the quiescent centre of Arabidopsis thaliana (Col.). J Exp Bot 44: 689–693[Abstract/Free Full Text] Gagliardi D, Gualberto JM (2004) Gene expression in higher plant mitochondria. In DA Fay, AH Millar, J Whelan, eds, Plant Mitochondria: From Genome to Function. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 55–82 Garg N, Gerstner A, Bhatia V, DeFord J, Papaconstantinou J (2004) Gene expression analysis in mitochondria from chagasic mice: alterations in specific metabolic pathways. Biochem J 381: 743–752[CrossRef][ISI][Medline] Geer BW, Krochko D, Oliver MJ, Walker VK, Williamson JH (1980) A comparative study of the NADP-malic enzymes from Drosophila and chick liver. Comp Biochem Physiol 65: 25–34[CrossRef] Giegé P, Hoffmann M, Binder S, Brennicke A (2000) RNA degradation buffers asymmetries of transcription in Arabidopsis mitochondria. EMBO Rep 1: 164–170[CrossRef][ISI][Medline] Giegé P, Sweetlove LJ, Cognat V, Leaver CJ (2005) Coordination of nuclear and mitochondrial genome expression during mitochondrial biogenesis in Arabidopsis. Plant Cell 17: 1497–1512[Abstract/Free Full Text] Grosskopf D, Mulligan RM (1996) Developmental- and tissue-specificity of RNA editing in mitochondria of suspension-cultured maize cells and seedlings. Curr Genet 29: 556–563[ISI][Medline] Hochholdinger F, Wulff D, Reuter K, Park WJ, Feix G (2000) Tissue specific expression of AUX1 in maize roots. J Plant Physiol 157: 315–319[ISI] Holness MJ, Sugden MC (2003) Regulation of pyruvate dehydrogenase complex activity by reversible phosphorylation. Biochem Soc Trans 31: 1143–1151[ISI][Medline] Hutter DE, Till BG, Greene JJ (1997) Redox state changes in density-dependent regulation of proliferation. Exp Cell Res 232: 435–438[CrossRef][ISI][Medline] Ivanov VB (2004) Meristem as a self-renewal system: maintenance and cessation of cell proliferation (a review). Russ J Plant Physiol 51: 834–847[CrossRef][ISI] Jiang K, Feldman LJ (2003) Root meristem establishment and maintenance: the role of auxin. J Plant Growth Regul 21: 432–440 Jiang K, Feldman LJ (2005) Regulation of root apical meristem development. Annu Rev Cell Dev Biol 21: 485–509[CrossRef][ISI][Medline] Jiang K, Meng YL, Feldman LJ (2003) Quiescent center formation in maize roots is associated with an auxin-regulated oxidizing environment. Development 130: 1429–1438[Abstract/Free Full Text] Jiang K, Zhang S, Lee S, Tsai G, Kim K, Huang H, Zhu T, Feldman LJ (2006) Transcription profile analyses identify genes and pathways central to root cap functions in maize. Plant Mol Biol 60: 343–363[CrossRef][ISI][Medline] Jiménez A, Hernández JA, Pastori G, del Río LA, Sevilla F (1998) Role of the ascorbate-glutathione cycle of mitochondria and peroxisomes in the senescence of pea leaves. Plant Physiol 118: 1327–1335[Abstract/Free Full Text] Lupold SD, Caoile AGFS, Stern DB (1999) Polyadenylation occurs at multiple sites in maize mitochondrial cox2 mRNA and is independent of editing status. Plant Cell 11: 1565–1578[Abstract/Free Full Text] Mancini M, Anderson BO, Caldwell E, Sedghinasab M, Paty PB, Hockenbery DM (1997) Mitochondrial proliferation and paradoxical membrane depolarization during terminal differentiation and apoptosis in a human colon carcinoma cell line. J Cell Biol 138: 449–469[Abstract/Free Full Text] Martin E, Rosenthal RE, Fiskum G (2005) Pyruvate dehydrogenase complex: metabolic link to ischemic brain injury and target of oxidative stress. J Neurosci Res 79: 240–247[CrossRef][ISI][Medline] McMaugh SJ, Lyon BR (2003) Real-time quantitative RT-PCR assay of gene expression in plant roots during fungal pathogenesis. Biotechniques 34: 982–986[ISI][Medline] Millar H, Considine MG, Day DA, Whelan J (2001) Unraveling the role of mitochondria during oxidative stress in plants. IUBMB Life 51: 201–205[ISI][Medline] Millenaar FF, Benschop JJ, Wagner AM, Lambers H (1998) The role of the alternative oxidase in stabilizing the in vivo reduction state in the ubiquinone pool and the activation state of the alternative oxidase. Plant Physiol 118: 599–607[Abstract/Free Full Text] Møller IM (2001) Plant mitochondria and oxidative stress: electron transport, NADPH turnover, and metabolism of reactive oxygen species. Annu Rev Plant Physiol Plant Mol Biol 52: 561–591[CrossRef][ISI][Medline] Nakajima Y, Mulligan MR (2001) Heat stress results in incomplete C-to-U editing of maize chloroplast mRNAs and correlates with changes in chloroplast transcription rate. Curr Genet 40: 209–213[CrossRef][ISI][Medline] Noda T, Iwakiri R, Fujimoto K, Aw TY (2001) Induction of mild intracellular redox imbalance inhibits proliferation of CaCo-2 cells. FASEB J 15: 2131–2139[Abstract/Free Full Text] Nulton-Persson AC, Szweda LI (2001) Modulation of mitochondrial function by hydrogen peroxide. J Biol Chem 276: 23357–23361[Abstract/Free Full Text] Nunes-Nesi A, Carrari F, Lytovchenko A, Smith AMO, Loureiro ME, Ratcliffe RG, Sweetlove LJ, Fernie AR (2005) Enhanced photosynthetic performance and growth as a consequence of decreasing mitochondrial malate dehydrogenase activity in transgenic tomato plants. Plant Physiol 137: 611–622[Abstract/Free Full Text] Pastori GM, del Río LA (1994) An activated oxygen-mediated role for peroxisomes in the mechanism of senescence of Pisum sativum L. leaves. Planta 193: 385–391[ISI] Randall DR (1982) Pyruvate dehydrogenase complex from broccoli and cauliflower. In WA Wood, ed, Carbohydrate Metabolism: Methods in Enzymology, Part D, Vol 89. Academic Press, New York, pp 408–414 Reichheld J-P, Vernoux T, Lardon F, Van Montagu M, Inzé D (1999) Specific checkpoints regulate plant cell cycle progression in response to oxidative stress. Plant J 17: 647–656[CrossRef][ISI] Smith J, Ladi E, Mayer-Pröschel M, Noble M (2000) Redox state is a central modulator of the balance between self-renewal and differentiation in a dividing glial precursor cell. Proc Natl Acad Sci USA 97: 10032–10037[Abstract/Free Full Text] Strain J, Lorenz CR, Bode J, Garland S, Smolen GA, Ta DT, Vickery LE, Culotta VC (1998) Suppressors of superoxide dismutase (SOD1) deficiency in Saccharomyces cerevisiae. J Biol Chem 273: 31138–31144[Abstract/Free Full Text] Sureau F, Moreau F, Millot JM, Manfait M, Allard B, Aubard J, Schwaller MA (1993) Microspectrofluorometry of the protonation state of ellipticine, an antitumor alkaloid, in single cells. Biophys J 65: 1767–1774[Abstract/Free Full Text] Sweet S, Singh G (1995) Accumulation of human promyelocytic leukemic (HL-60) cells at two energetic cell cycle checkpoints. Cancer Res 55: 5164–5167[Abstract/Free Full Text] Sweetlove LJ, Heazlewood JL, Herald V, Holtzapffel R, Day DA, Leaver CJ, Millar AH (2002) The impact of oxidative stress on Arabidopsis mitochondria. Plant J 32: 891–904[CrossRef][ISI][Medline] Valls V, Castelluccio C, Fato R, Genova ML, Bovina C, Saez G, Marchetti MG, Parenti Castelli G, Lenaz G (1994) Protective effect of exogenous coenzyme q against damage by adriamycin in perfused rat liver. Biochem Mol Biol Int 33: 633–642[ISI][Medline] Waterhouse NJ, Goldstein JC, von Ahsen O, Schuler M, Newmeyer DD, Green DR (2001) Cytochrome c maintains mitochondrial transmembrane potential and ATP generation after outer mitochondrial membrane permeabilization during the apoptotic process. J Cell Biol 153: 319–328[Abstract/Free Full Text] Williams MA, Tallakson WA, Phreaner CG, Mulligan RM (1998) Editing and translation of ribosomal protein S13 transcripts: unedited translation products are not detectable in maize mitochondria. Curr Genet 34: 221–226[CrossRef][ISI][Medline] Yoon Y-S, Lee J-H, Hwang S-C, Choi KS, Yoon G (2005) TGF beta1 induces prolonged mitochondrial ROS generation through decreased complex IV activity with senescent arrest in Mv1Lu cells. Oncogene 24: 1895–1903[CrossRef][ISI][Medline]
Related articles in Plant Physiol.:
- On the Inside
- Peter V. Minorsky
Plant Physiol. 2006 140: 791-792.
[Full Text]
This article has been cited by other articles:
|
|
|
|
 
K. Jiang, C. Schwarzer, E. Lally, S. Zhang, S. Ruzin, T. Machen, S. J. Remington, and L. Feldman
Expression and Characterization of a Redox-Sensing Green Fluorescent Protein (Reduction-Oxidation-Sensitive Green Fluorescent Protein) in Arabidopsis
Plant Physiology,
June 1, 2006;
141(2):
397 - 403.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
TOP
ABSTRACT
RESULTS