Molecular Plant Advance Access originally published online on June 27, 2008
Molecular Plant 2008 1(4):675-685; doi:10.1093/mp/ssn031
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A Mitochondrial Magnesium Transporter Functions in Arabidopsis Pollen Development
a College of Life Sciences, Capital Normal University, Beijing 100037, China
b Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA
c NJU–NJFU Joint Institute for Plant Molecular Biology, College of life Sciences, Nanjing University, Nanjing 210093, China
d College of Life Sciences, Hunan Normal University, Changsha 410006, China
1 To whom correspondence should be addressed. E-mail sluan{at}nature.berkeley.edu lgli11242006{at}gmail.com, fax (510) 642-4995 or 86–10–68981191, tel. (510) 642-6306 or 86–10–68902593.
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Abstract |
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Magnesium is an abundant divalent cation in plant cells and plays a critical role in many physiological processes. We have previously described the identification of a 10-member Arabidopsis gene family encoding putative magnesium transport (MGT) proteins. Here, we report that a member of the MGT family, AtMGT5, functions as a dual-functional Mg-transporter that operates in a concentration-dependent manner, namely it serves as a Mg-importer at micromolar levels and facilitates the efflux in the millimolar range. The AtMGT5 protein is localized in the mitochondria, suggesting that AtMGT5 mediates Mg-trafficking between the cytosol and mitochondria. The AtMGT5 gene was exclusively expressed in anthers at early stages of flower development. Examination of two independent T-DNA insertional mutants of AtMGT5 gene demonstrated that AtMGT5 played an essential role for pollen development and male fertility. This study suggests a critical role for Mg2+ transport between cytosol and mitochondria in male gametogenesis in plants.
Key Words: Mg2+-transporter • pollen • mitochondria • Arabidopsis
Received for publication February 29, 2008. Accepted for publication April 4, 2008.
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INTRODUCTION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSFUNDING |
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Magnesium (Mg2+) is the most abundant divalent cation in living cells, and is essential for a wide range of cellular functions in all organisms. Being the central metal of the chlorophyll molecule, Mg2+ is critically important for photosynthesis in higher plants. Additionally, it serves as a co-factor for many enzymes, including RNA polymerases, ATPases, protein kinases, and phosphatases that play a role in many cellular processes. Intracellular Mg2+ concentrations are precisely regulated by transporters that mediate Mg2+ uptake, sequestration into cellular storage compartments, and Mg2+ efflux (Shaul, 2002; Gardner, 2003). Earlier studies in bacteria, especially in Salmonella typhimurium, identified two major Mg-transporters (Hmiel et al., 1986; Snavely et al., 1989). The MgtA/B belong to ATPase-type transporters (Hmiel et al., 1989; Smith et al., 1993) and CorA protein represents a novel type of transporter with a large, acidic, N-terminal, periplasmic domain and only two transmembrane domains at the C-terminal region. Following the bacterial studies, a number of studies have shown that CorA-like proteins may represent the major transport systems in other organisms including eukaryotes such as yeast, animals, and plants (Bui et al., 1999; Graschopf et al., 2001; Kolisek et al., 2003; Schock et al., 2000; Li et al., 2001). For example, a yeast mutant defective in mitochondrial RNA splicing has been shown to be mutated in a CorA-related gene called MRS2 (mitochondrial RNA Splicing 2). A study has confirmed that MRS2 indeed encodes a functional Mg-transporter (Bui et al., 1999). In an attempt to identifying aluminum resistance genes, researchers isolated ALR1/ALR2 genes encoding CorA-like proteins from yeast, linking Al toxicity to Mg-transport (Macdiarmid and Gardner, 1998). Indeed, both ALR1 and ALR2 can transport Mg2+ in yeast cells (Graschopf et al., 2001; Liu et al., 2002). The CorA-like proteins have also been identified from animals and human (Kolisek et al., 2003; Zsurka et al., 2001). The human Mrs2 protein can functionally substitute for its yeast homolog. Most of the CorA-like genes in mammals belong to MRS2-type proteins that may be located in the mitochondria (Kolisek et al., 2003; Zsurka et al., 2001). It remains unknown if CorA-like transporters also exist in the plasma membrane in animal cells.
Two independent studies identified a 10-member gene family encoding CorA-like magnesium transporters in Arabidopsis (Schock et al., 2000; Li et al., 2001). The study by Schock et al. (2000) isolated one of the family members as a plasma membrane-localized protein that complements the yeast mrs2 mutant and named the gene family AtMRS2. Study by Li et al. (2001) showed that two family members complemented defects in magnesium transport in a bacterial and yeast mutant, respectively, and named the gene family MGT (Mg-Transport). In addition, the two MGT members expressed in the bacterial and yeast cells were shown to uptake magnesium from the medium. In particular, AtMGT1 appeared to transport Mg with a high affinity comparable to the Mg-affinity of CorA protein in bacteria (Li et al., 2001). Although these studies provide strong evidence that AtMRS2/AtMGT genes encode magnesium transporters in Arabidopsis, their in-planta physiological functions remain unknown. To understand the in-vivo function of AtMGT family members, we combined the bacterial transport model system and Arabidopsis genetic analysis to determine both the Mg-transporting activity and the in-vivo function of individual AtMGT members. Here, we show that AtMGT5 serves as a dual-functional transporter that mediates high-affinity Mg2+ uptake under micro-molar [Mg] conditions and facilitates Mg2+ efflux when the outside assay [Mg] increases to millimolar levels. The AtMGT5 protein is localized in the mitochondria and is specifically expressed in the anther tissues during flower development. Our genetic analyses demonstrated that AtMGT5 function is essential for pollen development.
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AtMGT5 Complements a Bacterial Strain Deficient in Mg2+ Uptake
The AtMRS2/AtMGT family consists of at least 10 rather divergent members (Schock et al., 2000; Li et al., 2001). Although they contain conserved regions found in all plant members and in homologs from other organisms ranging from bacteria to human, the overall sequence similarity among the members is considerably lower. Based on sequence homology, the AtMGT family can be divided into four subfamilies. Members from AtMGT1 to AtMGT4 belong to subgroup I, AtMGT5 and AtMGT6 are grouped in II, AtMGT7, 8, and 9 form subgroup III, and 10 is very different from all the rest and stands alone as subgroup IV (Li et al., 2001). The members in the same subfamily are highly related in sequence and structure, and could function similarly. The functionality of these transporters can be reflected by their transporting activity, including substrate specificity and affinity, and their functions in various physiological processes in plants. For example, our earlier study (Li et al., 2001) showed that AtMGT1 has a function as a high-affinity Mg-transporter in plant cells like the CorA protein in bacteria. We speculated that other members in the same subfamily such as AtMGT2/AtMRS2-1 may have a similar function as AtMGT1 for Mg2+ transport. In contrast, members from other subfamilies may be different regarding their transport activity and physiological functions. In an effort to test this idea and to characterize the function of all AtMGT members, we cloned AtMGT5, a member from subfamily II, into the bacterial expression vector and examined its transport activity in the MM281 bacterial strain lacking Mg-transporting systems (Hmiel et al., 1989; Li et al., 2001; Kehres and Maguire, 2002).
The MM281 cells do not survive in the low-Mg medium (<1 mM) at pH 7.5, and functional Mg-transporters such as CorA and AtMGT1 can readily complement this defect and rescue the growth of the MM281 mutant on medium containing as little as 1 mM Mg (Li et al., 2001). Using the same complementation assay, we tested if AtMGT5 could function as a Mg-transporter. As shown in Figure 1A, the growth of MM281 cells in the low-Mg medium was restored after transformation with AtMGT5 cDNA (AtMGT5-MM281). However, we noted that growth of AtMGT5-MM281 cells was slower, compared with the strain expressing AtMGT10, a member that showed Mg-transport activity in yeast cells (Li et al., 2001). We also compared AtMGT1, AtMGT10, and AtMGT5 and found that AtMGT10 activity in the functional complementation assay is comparable to AtMGT1 but AtMGT5 is less effective in the complementation (data not shown). This suggests that AtMGT5 could mediate Mg-uptake but may not be as effective an Mg-transporter as AtMGT10 or AtMGT1. To further examine the complementation of MM281 strain by AtMGT5, we monitored the cell growth in the liquid culture and plotted a growth curve of several strains, including MM281, and MM281 transformed with AtMGT10 or AtMGT5 expression constructs. This experiment confirmed that MM281 carrying AtMGT5 grew well as compared to the non-complemented mutant strain but lagged behind the AtMGT10-complemented strain in the low-Mg medium (Figure 1B).
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AtMGT5 Is a Dual-Functional Mg-Transporter that Mediates Concentration-Dependent Influx and Efflux
In the complementation tests, we noted that AtMGT5-transformed cells grew well under low [Mg2+] but did not grow when the [Mg2+] in the medium reached millimolar levels (Figure 1A). This result is puzzling because cells transformed with other members of the MGT family, such as AtMGT1 or AtMGT10, grew well under both low- and high-Mg2+ concentrations. One plausible hypothesis is that AtMGT5 may mediate Mg2+ efflux under high-[Mg2+] conditions, thereby inhibiting bacterial growth. To test this hypothesis, we performed tracer uptake assay as previously described (Li et al., 2001). This assay utilized the fact that Mg2+ and Ni2+ are transported by the same transporters with similar kinetics (Szegedy and Maguire, 1999). Inhibition of Ni2+ tracer uptake by non-radioactive Mg2+ or other cations represents the uptake efficiency of Mg2+ or other cations. The results depicted in Figure 2 indicate that the uptake kinetics for Ni2+ and Mg2+ displayed a similar pattern in MM281 cells. In the mutant strain, Ni2+ and Mg2+ began to inhibit tracer uptake at 1 mM concentration, which is consistent with the fact that MM281 required more than 1 mM MgSO4 to grow. AtMGT5-transformed cells began to show inhibition of tracer uptake when Mg2+ concentration reached 0.1 mM, as indicated in Figure 2A. Interestingly, at 10 mM, the inhibition of uptake is saturated. In other words, the inhibition of tracer uptake is not significantly increased as the Mg concentration increased from 10 to 1 mM, in sharp contrast with the pattern of AtMGT10 (or AtMGT1) uptake (Figure 2A; also see ref. Li et al., 2001). Interestingly, the inhibition of tracer uptake by Ni is concentration-dependent and positively correlated with the Ni concentration, which is consistent with the pattern of AtMGT10. This result indicated that AtMGT5-mediated influx is specifically inhibited by a high concentration of magnesium (Figure 2B). This could suggest that AtMGT5 may mediate Mg2+ influx under low-Mg2+ concentrations but its transport property is altered when extracellular Mg2+ concentrations increase to high micromolar and millimolar levels.
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Does AtMGT5 mediate Mg2+ efflux under high Mg2+ conditions? We tested this idea by efflux assay. Bacterial cells were first loaded with 63Ni2+ as described for uptake assay, followed by washing the excess isotope with ice water. The aliquots of cells with equal amount of radioactivity were transferred in the efflux assay buffer containing different Mg2+ concentrations and incubated at 37°C for 30 min. The cells were then precipitated and washed before determining the remaining isotope activity in the cells. Figure 3A indicated that the amount of 63Ni2+ in the MM281 and MM281-AtMGT10 cells did not change significantly after efflux assay in the presence of different Mg2+ concentrations. However, 63Ni2+ in the cells containing AtMGT5 dramatically decreased as magnesium concentration increased to 0.1 mM and higher. As shown in Figure 3B, we measured the efflux rates at different time points with 10 mM MgCl2 in the external medium. Under this condition, the efflux rate of AtMGT5 cells was much higher than MM281 or AtMGT10 cells.
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To further investigate the functional properties of AtMGT5, we performed tracer inhibition assays with a number of other divalent cations to determine the ionic selectivity of AtMGT5. Several divalent cations, including Cu2+, Mn2+, and Co2+, significantly inhibited tracer uptake in AtMGT5-transformed cells. The concentrations required for 50% inhibition were in the range of 100 uM to 10 mM for Cu2+, Mn2+, and Co2+, which are beyond the physiological ranges of these cations. In addition, the kinetics of inhibition was similar to that in MM281 mutant cells, ruling out a possible role of AtMGT5 in the uptake of these cations under physiological conditions (data not shown). These results indicate that AtMGT5 may function specifically as a Mg2+ transporter in plants.
In this study, we also show that AtMGT10 shares similar transport properties with AtMGT1 published previously (Li et al., 2001), whereas AtMGT5 transport activity is significantly different. The difference was not only reflected in the kinetics of tracer uptake assay, but also in the sensitivity to aluminum. As shown earlier (Li et al., 2001), the uptake by AtMGT1 and AtMGT10 was strongly inhibited by Al. However, AtMGT5 was not sensitive to Al (data not shown). Our results revealed that the AtMGT members of different subfamilies may have different functional properties in maintaining the ion homeostasis.
AtMGT5 Is Localized in the Mitochondria
Besides the transport activity, sub-cellular localization is also a critical factor that will determine the in-vivo function of a given AtMGT member. Earlier studies showed that subtype-I members AtMGT1 and MRS2-1/AtMGT2 are both localized to the plasma membrane of plant cells (Schock et al., 2000; Li et al., 2001). We examined the sub-cellular localization of AtMGT5 protein, a subtype-II that serves as a dual-functional Mg2+ transporter. Using the transgenic plants expressing GFP–AtMGT5 fusion protein, we localized the GFP fluorescence by confocal microscopy (see Methods). Because the expression of GFP–AtMGT5 was driven by the 35S promoter, we focused on the chlorophyll-free tissues such as roots to avoid interference from auto-fluorescence. Unlike GFP–AtMGT1 that is associated with the cell periphery (Li et al., 2001), GFP–AtMGT5 was detected in intracellular organelles that rapidly move inside the cytoplasm as indicated by changes in the position of fluorescence signals in the same cell in different scanning profiles (Figure 4A and 4B, and movie—not shown). Based on the size, number, and behavior of the GFP-harboring organelles, we considered them to be mitochondria. We used MitoTracker Red CMXRos fluorescence to localize mitochondria and found that GFP signals completely overlapped with the Mitotracker Red (Figure 4C–4E), indicating that AtMGT5 protein is localized to the mitochondria in planta.
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AtMGT5 Gene Is Specifically Expressed in Anther Tissues during Early Flower Development
We previously noticed that AtMGT5 was expressed in flowers but not in leaves, stems, or roots (Li et al., 2001). To study the physiological function of AtMGT5, we further examined the expression pattern of AtMGT5 gene at various stages of flower development and in different floral organs using RT–PCR. Figure 5 shows that AtMGT5 gene transcripts were accumulated at early stages of flower development, approximately corresponding to flower stages from 9 to 13 as defined by Smyth et al. (1990). The AtMGT5 transcript became undetectable in open flowers. When different floral organs were collected from early stages and analyzed for AtMGT5 expression, AtMGT5 transcripts were detected only in the anther (Figure 5A).
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To conduct further histological analysis of AtMGT5 expression pattern, we made a reporter gene construct containing the promoter region of AtMGT5 gene driving expression of GUS gene and produced transgenic plants containing this construct. Analysis of GUS activity in the transgenic plants indicated that AtMGT5 promoter was not active in the vegetative tissues (not shown). When flowers of different stages were stained, GUS activity was specifically detected in anthers of early-stage flower buds (Figure 5B). As the flower buds developed further to open flowers, the GUS activity disappeared, consistent with the RT–PCR analysis of the native AtMGT5 mRNA in Figure 5A.
To determine the cell types that express AtMGT5 gene, we sectioned the GUS stained flower buds and examined GUS staining pattern inside the anther. A rather unique temporal and spatial pattern of GUS activity was observed. The onset of AtMGT5 promoter activity began as early as stages 4–5 of pollen development as defined by Sanders et al (Figure 5B: d, f, g; Sanders et al., 1999). The GUS accumulation at this stage is evenly distributed throughout the anther (Figure 5B: b, c). With the progression of the pollen/anther development, from pollen developmental stages 6–9, AtMGT5 promoter activity was preferentially distributed in the vascular region and connective tissues of anthers, although it was not restricted to those tissues (Figure 5B: f, g). This increased promoter activity was particularly prominent at the junction of the filament and the anther (Figure 5B: f, g, and data not shown). AtMGT5 expression was also detected to be associated with Arabidopsis pollen grains throughout these stages (pollen development stages 6–9: Figure 5B: f, i, h). At stages 9 and 10, in the onset of the pollen maturation, the spatial pattern of AtMGT5 expression is shifted to tapetum, the ‘nursing tissue’ of pollen development and the promoter remains active also in the vascular tissues. Interestingly, at the stage of opening the septum between the loculi pairs (stage 12), when the tapetum tissue is degraded, there is still activity present in the vascular tissues and occasionally associated with pollen grains (Figure 5B: k). Taken together, these results illustrate that the expression of AtMGT5 gene appears to ‘shift’ from the filament–anther junction to the tapetum, and eventually to pollen grains along the developmental progression of microspores.
AtMGT5 Is Essential for Pollen Development
The AtMGT5 gene is expressed in both gametophytic and sporophytic tissues in a manner coordinated spatially and temporally with pollen development (Figure 5). We speculated that AtMGT5 may, therefore, play a critical role in the formation of the microspores. To test this idea and address the physiological significance of AtMGT5 function, we took a reverse genetics approach to examine the consequence of disrupting this gene in Arabidopsis (Winkler and Feldmann, 1998; Krysan et al., 1999). Two T-DNA insertional mutants of the AtMGT5 gene were isolated from Syngenta and ABRC collections of T-DNA-transformed Arabidopsis lines (allele-1, allele-2). The insertional sites were mapped to the –147 and +968 in allele 1 and 2, respectively, in relation to the first nucleotide (+1) of the translation start codon (ATG) (Figure 6A). When we attempted to produce homozygous lines of these mutants, we never succeeded with either allele (out of a pool of 100 progeny individual plants per screen). This result suggests that AtMGT5 may be essential for gametophytes or/and embryo development. The anther-specific expression pattern of the AtMGT5 gene strongly suggests that AtMGT5 mutants may have defects in the development of male gamete (microspore). We examined the pollen grains in the heterozygous mutant flowers and indeed found that about 50% of pollens collapsed at stages close to maturation (Figure 6B: b and 6C: c, f), consistent with the ratio of vital and dead pollen grains typical for heterozygous gemetophytic lethal mutants. The high percentage of dead pollen grains in the heterozygous plants correlated with reduced silique size in these plants (data not shown). The defects of pollen grains were examined more closely using environmental SEM. Anthers and pollen grains from control plants appeared normal (Figures 6C: a, d), whereas a number of abnormal and collapsed pollen grains were apparent in the anthers from heterozygous mutant plants (Figures 6C: b, c, d, e). The defective pollen morphology and percentage of aborted pollen grains were consistent with the pollen grain deformations observed using brightfield microscopy (Figure 6B: b) and with the death rate analyzed using DAPI-staining and UV-fluorescence microscopy (Figure 6B: c).
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DISCUSSION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSFUNDING |
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Despite the importance of Mg2+ in plant physiology, little is understood on the acquisition, transport, and cellular homeostasis of this cation due to poor understanding of the transport systems. We recently reported identification of a multigene family encoding Mg2+ transporters in Arabidopsis (AtMGT), setting the stage for dissecting the functional significance of the Mg2+ transport systems in plant cells. Our study here identified a physiological function of a novel MGT member that displays unique property as a dual-functional transporter localized in the mitochondrial membrane. The transport activity mediates both influx and efflux of Mg2+ in a mode of action that is switched by the external Mg2+ concentrations. The expression of this novel transporter is specifically associated with the male gametophyte and tightly regulated by developmental stage. Consistent with its expression pattern, activity of AtMGT5 is essential for male gametophyte vitality. Our study provides genetic evidence on an intimate relationship between mitochondrial Mg2+ transport and male gamete development.
The AtMGT5 transporter is unique as compared to the AtMGT1 and AtMGT10 studied earlier in two major aspects. First, the AtMGT1 is a high-affinity influx transporter that is not regulated by Mg2+ concentration whereas AtMGT5 mediates both influx and efflux of Mg2+, depending on the external [Mg2+]. In the tracer uptake assay, the uptake activity of AtMGT1 and AtMGT10 increased throughout the tested [Mg2+] range (0.1 uM to 10 mM). However, uptake activity of AtMGT5 does not increase beyond 10 uM external [Mg2+]. In addition, the efflux assay revealed that AtMGT5 began to function as an efflux carrier as the Mg concentration reached the high uM to mM range when the ‘uptake’ function of this transporter is terminated. Such switch may mimic the voltage-independent cation channels that mediate ionic current across the membrane, depending on the concentration (and electrochemical gradient across the membrane). However, the unique property of AtMGT5 is opposite to those channels in that its efflux activity is activated by higher concentration on the external side of the membrane. The intriguing property of this transporter may be related to the second major difference between AtMGT1 and AtMGT5. The AtMGT1 is located to the plasma membrane whereas AtMGT5 is localized in the mitochondria. The [Mg2+]-dependent activity of AtMGT5 may be important for the homeostasis of the Mg between mitochondria and cytosol. When the concentration of Mg in the cytosol is in the low micromolar range, the AtMGT5 serves as an uptake transporter to maintain Mg2+ in the mitochondria. However, when the cytosolic concentration reached millimolar range, the transporter will no longer function as an uptake transporter and instead it serves as an efflux transporter to prevent Mg2+ ‘flooding’ into the mitochondria. It is possible that at high cytosolic Mg2+ concentrations, other uptake transporter(s) are activated to keep the influx pathway open for homeostatic control. Such a Mg2+-concentration-dependent transporter has not been reported in other systems and may represent a unique mechanism for mitochondria–cytosol interaction in plant cells. For example, the AtMGT5 may serve as a mitochondrial ‘sensor’ for the cytosolic Mg levels and thereby functioning in the homeostasis of this regulatory cation.
The importance of mitochondrial cytosolic Mg2+ transport is clear when we consider the function of Mg2+ inside the mitochondria. As Mg2+ is a critical cofactor of ATP synthases and other enzymes in the respiration processes, Mg2+ level should serve as an essential regulatory element of mitochondrial activity. As mitochondrial defects have long been implicated in male sterility, it is not fortuitous that AtMGT5 plays an essential role in pollen development. One particular case of intensive interest is cytoplasmic male sterility (CMS) caused by defects in the mitochondrial ATP synthase gene. Mitochondrial loci correlated with CMS were first identified in maize and almost all of them are related to ATP synthase subunits (Cui et al., 1996; Sabar et al., 2003; Heazlewood et al., 2003; Hanson and Bentolila, 2004). Whatever defect in mitochondrial function is caused by a CMS-associated region, its ultimate result may be programmed cell death (PCD) of either the tapetal layer or sporogenous cells. During normal development, it has been shown that PCD takes place in some anther sporophytic tissues, resulting in tapetum disappearance and development of the dehiscence zone (Clément et al., 1998; Papini et al., 1999; Schreiber et al., 2004). Even before PCD was recognized as a specific programmed process, many investigators documented microscopic evidence of premature breakdown of the tapetal layer and mitochondrial morphology changes in dying anther cells (He et al., 1996; Horner, 1977). As in animals, plant mitochondria appear to play a role in PCD. Again, the AtMGT5 may be important in the function of tapetum cells, as its gene is expressed in such cells during early pollen development. Although further experiments are required to test whether AtMGT5 indeed mediates Mg transport between cytosol and mitochondria, it is tempting to hypothesize that precise control of Mg2+ concentration is crucial for mitochondrial activity and AtMGT5 plays a critical role in this control mechanism. Because mitochondrial function is a key element for cytosolic male fertility, disruption of AtMGT5 (and Mg transport) thus causes male sterility through aberration in pollen development.
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AtMGT5 cDNA Cloning and Salmonella typhimurium Mutant Complementation
The S. typhimurium mutant MM281, which lacks CorA, MgtA, and MgtB genes (Smith et al., 1995), was used in the complementation study. AtMGT5 cDNA was cloned and inserted into a pTrc99A vector as previously reported (Li et al., 2001). MM281 cells were transformed with pTrc99A vector and pTrc99A–AtMGT5 plasmid by electroporation. Cells were plated onto LB medium supplemented with 20 mM Mg2+ and appropriate antibiotics (100 mg ml–1 ampicillin, 34 mg ml–1 chloramphenicol, and 50 mg ml–1 kanamycin), and incubated at 37°C overnight. The transformants were confirmed by PCR amplification of both the vector and AtMGT5 coding sequences. Individual transformants were grown in LB liquid medium containing the same concentrations of Mg2+, antibiotics and 0.05 mM IPTG for induction of AtMGT5 expression. MM281-MGT10 was used as a positive control. The cultures were adjusted to 1.0 OD600, diluted in a 10-fold series, and spotted (2 ml) onto N-minimal medium without Tris-HCl supplemented with different concentrations of MgSO4 and the antibiotics (Nelson and Kennedy, 1971). Growth of different strains was scored after incubation at 37°C for 24 h.
Tracer Uptake Assays
Uptake of 63Ni was performed as described previously (Snavely et al., 1989; Smith et al., 1998). 63Ni uptake was assayed using bacterial strain MM281 containing the pTrc99A empty vector or MGT10, AtMGT5 expression construct. Bacterial cultures were grown overnight in LB medium containing 20 mM Mg2+ and the appropriate antibiotics. When the concentration of the cells reached OD600 = 1, 0.05 mM IPTG was added to the culture medium and the culture was grown for five additional hours. Cells were washed with N-minimal medium without Tris-HCl and Mg2+, and diluted 1:5 with the same medium containing appropriate antibotics. After a 3-h subculture in the minimal medium, cells were collected by centrifugation at 1000 g for 15 min and washed twice with ice-cold Mg2+-free N-minimal medium. The washed cells were resuspended in the same medium and cell density was adjusted to 1.0 OD600. For a standard assay, uptake was initiated by adding 0.2 mL cells to 0.8 mL of N-minimal medium containing 100 mM NiCl2 and 0.5 mCi of 63Ni2+. For the ionic selectivity assay, various concentrations of divalent cations were included in the tracer uptake buffer. Typically, uptake was stopped after 5 min by adding 1 mL of ice-cold washing buffer. Cells were washed four times with 1.5 mL of ice-cold washing buffer and the 63Ni2+ content of bacterial cells was determined by a scintillation counter (Beckman, LS600IC). The relative tracer uptake was standardized against the maximal value and presented as per cent of maximal uptake.
Plant Material and RT–PCR Procedures
To analyze AtMGT5 mRNA expression pattern, total RNA was extracted from flower clusters of different developmental stages, and from different floral organs including sepal, petal, anther, ovary, flower base, and silique, with RNAeasy Kit (Qiagen, Santa Clarita, CA). Total RNA (2.5 mg) was heated at 65°C for 10 min and then subjected to reverse transcription (RT) reaction using SuperTranscriptase II (Invitrogen, Carlsbad, CA) and oligo dT18 for 60 min at 42°C. 1 ml of RT reaction was used as a template to amplify AtMGT5 with AmpliTaq (Perkin Elmer Branchburg, NJ). The amplifications were performed with initial denaturation at 94°C for 3 min followed by 30 standard PCR reaction cycles, and a final extension at 72°C for 10 min. Arabidopsis ACTIN3 gene was used as a quantification control. Aliquots of PCR samples were resolved by agarose gel electrophoresis and visualized by ethidium bromide staining under UV light in a UVP BioDocItTM System (UVP, Inc. Upland, CA).
Sub-Cellular Localization of AtMGT5–GFP Fusion Protein
The AtMGT5 coding region without the stop codon was fused to the N-terminus of GFP coding region in binary vector pMD1 that contains the CMV 35S promoter followed by a short polylinker, GFP-coding region, and the NOS terminator region. This construct was used to transform wild-type Arabidopsis plants (Columbia-0 ecotype) by vacuum infiltration (Bechtold et al., 1993). Plants from T3 seeds were used to localize GFP by a confocal microscope (Zeiss 510 UV/Vis) as previously described (Li et al., 2001). MitoTracker Red CMXRos (Invitrogen, Carlsbad, CA) was used to stain mitochondria of the same materials used for GFP analysis. The dye was dissolved in dimmethyl sulfoxide to 1 mM stock solution and diluted into a final concentration of 0.5 uM for use in the staining procedure described by the manufacturer.
Construction of the AtMGT5 Promoter β-Glucuronidase Fusion (AtMGT5-GUS) and Analysis of Transgenic Plants
A 1.8-kb fragment containing the sequence upstream of the –ATG start codon of AtMGT5 was amplified by PCR using AtMGT5PF (5'-GCTCTAGAATGTAAAGTTAATATTTACGTGACTAT) and AtMGT5PR (5'-CGGGATCCCATTGACTTACCTGCAGCTGGTCAGAGA) primers. The amplified AtMGT5 promoter was cloned as a translational fusion with the beta-glucuronidase (GUS) coding sequence replacing the AtMGT5 coding region in the plant binary vector pBI101.1 (Datla et al., 1992; Jefferson et al., 1986). Transformed Arabidopsis plants carrying the promoter–GUS fusion were selected based on Kanamycin resistance. The promoter activity was visualized in the plant tissues by the GUS activity using 5-bromo-4-chloro-3-indolyl-β-D-glucuronide as a substrate according to published protocols (Jefferson et al., 1987). For further analysis of the GUS staining pattern, GUS-stained flower clusters were embedded in paraffin, sectioned and examined under microscope. For paraffin embedding, GUS-stained flowers were gradually dehydrated and cleared in progressively increasing ethanol concentrations up to 100% EtOH. The 100% ethanol step was repeated three times. The fixation of samples was continued in HistoclearTM (AGTC Bioproducts Ltd, UK) and EtOH solutions at room temperature for 3 h. Tissue was fixed by substituting HistoclearTM (AGTC Bioproducts Ltd, UK) for ethanol until 100% Histoclear was reached (i.e. 1:4, 1:1, 3:4 Histoclear:EtOH (vol:vol)). The final 100% Histoclear fixation step was repeated once. All fixation volumes (Histoclear/Ethanol) were ensured to be in at least 25 volumes excess to the inflorescence volume. Molten paraffin, heated at 60°C (Fisher Scientific, USA), was gradually substituted for the HistoclearTM solvent in the following increments: 1:3, 1:1, 3:4, and finally the inflorescence samples were embedded in 100% molten paraffin in moderately heated aluminum trays to allow paraffin solidification. Paraffin chips containing GUS-stained inflorescences were excised and mounted on wooden bases, sectioned at 8–10 mm, and mounted onto glass microscope slides with adhesive and de-waxed in xylene according to Ruzin (1999). An Axiophot 373 microscope was used to examine the sectioned samples.
Isolation of AtMGT5 Knock-Out Alleles and Phenotypic Analysis
T-DNA insertions within the AtMGT5 gene were identified using a primer specific for the T-DNA left border and AtMGT5-specific primers (allele-1: Garlic 562_G08 was provided by Syngenta; allele-2: SALK_037061 was from ABRC). The locations and orientation of the primers are shown in Figure 6A.
Pollen grains from the plants harboring the heterozygous T-DNA alleles were collected and analyzed using brightfield, flourescent (Axiophot 373) and environmental scanning electron microscopy (SEM) as described previously (Gupta et al., 2002).
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSFUNDING |
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This work is supported, in part, by grants from NSFC (30771077) (to L.L.) and the Hunan Young Scientist Program (03JJY1003) and National Science Foundation (to S.L.).
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Acknowledgements |
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We thank Dr Michael E. Maguire for providing bacterial strains and advice on tracer uptake assays and Drs Steven Ruzin and Denise Schichnes for technical advice on the inflorescence fixation and embedding protocol.
No conflict of interest declared.
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Notes |
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2 These authors contributed equally to this work.
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Bechtold N, Ellis J, Pelletier G. In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C.R. Acad. Sci. Paris Life Sci. (1993) 316:1194–1199.
Bui DM, Gregan J, Jarosch E, Ragnini A, Schweyen RJ. The bacterial magnesium transporter CorA can functionally substitute for its putative homologue Mrs2p in the yeast inner mitochondrial membrane. J. Biol. Chem. (1999) 274:20438–20443.
Clément C, Laporte P, Audran JC. The loculus content and tapetum during pollen development in Lilium. Sex. Plant Reprod. (1998) 11:94–106.[CrossRef]
Cui X, Wise RP, Schnable PS. The rf2 nuclear restorer gene of male-sterile T-cytoplasm maize. Science. (1996) 272:1334–1336.[Abstract]
Datla RS, Hammerlindl JK, Panchuk B, Pelcher LE, Keller W. Modified binary plant transformation vectors with the wild-type gene encoding NPTII. Gene. (1992) 122:383–384.[CrossRef][Web of Science][Medline]
Gardner RC. Genes for magnesium transport. Curr. Opin. Plant Biol. (2003) 6:263–267.[CrossRef][Web of Science][Medline]
Graschopf A, Stadler JA, Hoellerer MK, Eder S, Sieghardt M, Kohlwein SD, Schweyen RJ. The yeast plasma membrane protein Alr1 controls Mg2+ homeostasis and is subject to Mg2+-dependent control of its synthesis and degradation. J. Biol. Chem. (2001) 276:16216–16222.
Gupta R, Ting JT, Sokolov LN, Johnson SA, Luan S. A tumor suppressor homolog, AtPTEN1, is essential for pollen development in Arabidopsis. Plant Cell. (2002) 14:2495–2507.
Hanson MR, Bentolila S. Interactions of mitochondrial and nuclear genes that affect male gametophyte development. Plant Cell. (2004) 16:S154–S169.
He S, Abad AR, Gelvin SB, Mackenzie SA. A cytoplasmic male sterility-associated mitochondrial protein causes pollen disruption in transgenic tobacco. Proc. Natl Acad. Sci. U S A. (1996) 93:1763–1768.
Heazlewood JL, Whelan J, Millar AH. The products of the mitochondrial orf25 and orfB genes are FO components in the plant F1FO ATP synthase. FEBS Lett. (2003) 540:201–205.[CrossRef][Web of Science][Medline]
Hmiel SP, Snavely MD, Florer JB, Maguire ME, Miller CG. Magnesium transport in Salmonella-typhimurium genetic characterization and cloning of three magnesium transport loci. J. Bacteriol. (1989) 171:4742–4751.
Hmiel SP, Snavely MD, Miller CG, Maguire ME. Magnesium transport in Salmonella-typhimurium characterization of magnesium influx and cloning of a transport gene. J. Bacteriol. (1986) 168:1444–1450.
Horner H. A comparative light- and electron-microscopic study of microsporogenesis in male-fertile and cytoplasmic male-sterile sunflower (Helianthus annuus). Am. J. Bot. (1977) 64:745–759.[CrossRef][Web of Science]
Jefferson RA, Burgess SM, Hirsh D. Beta Glucuronidase from Escherichia Coli. as a gene-fusion marker. Proc. Natl Acad. Sci. U S A. (1986) 83:8447–8451.
Jefferson RA, Kavanagh TA, Bevan MW. Gus fusions Beta Glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. (1987) 6:3901–3908.[Web of Science][Medline]
Kehres DG, Maguire ME. Structure, properties and regulation of magnesium transport proteins. BioMetals. (2002) 15:261–270.[CrossRef][Web of Science][Medline]
Kolisek M, Zsurka G, Samaj J, Weghuber J, Schweyen RJ, Schweigel M. Mrs2p is an essential component of the major electrophoretic Mg2+ influx system in mitochondria. EMBO J. (2003) 22:1235–1244.[CrossRef][Web of Science][Medline]
Krysan PJ, Young JC, Sussman MR. T-DNA as an insertional mutagen in Arabidopsis. Plant Cell. (1999) 11:2283–2290.
Li L, Tutone AF, Drummond RSM, Gardner RC, Luan S. A novel family of magnesium transport genes in Arabidopsis. Plant Cell. (2001) 13:2761–2775.
Liu GJ, Martin DK, Gardner RC, Ryan PR. Large Mg(2+)-dependent currents are associated with the increased expression of ALR1 in Saccharomyces cerevisiae. FEMS Microbiol. Lett. (2002) 213:231–237.[CrossRef][Web of Science][Medline]
Macdiarmid CW, Gardner RC. Overexpression of the Saccharomyces cerevisiae magnesium transport system confers resistance to aluminum ion. J. Biol. Chem. (1998) 273:1727–1732.
Nelson DL, Kennedy EP. Magnesium transport in Escherichia coli. Inhibition by cobaltous ion. J. Biol. Chem. (1971) 246:3042–3049.
Papini A, Mosti S, Brighigna L. Programmed-cell-death events during tapetum development of angiosperms. Protoplasma. (1999) 207:213–221.[CrossRef][Web of Science]
Ruzin SE. Plant Microtechnique & Microscopy (Oxford University Press) (1999) 61–85.
Sabar M, Gagliardi D, Balk J, Leaver CJ. ORFB is a subunit of F1F(O)–ATP synthase: insight into the basis of cytoplasmic male sterility in sunflower. EMBO Rep. (2003) 4:381–386.[CrossRef][Web of Science][Medline]
Sanders PM, Bui AQ, Weterings K, McIntire KN, Hsu YC, Lee PY, Truong MT, Beals TP, Goldberg RB. Anther developmental defects in Arabidopsis thaliana male-sterile mutants. Sex. Plant Reprod. (1999) 11:297–322.[CrossRef]
Schock I, Gregan J, Steinhauser S, Schweyen R, Brennicke A, Knoop V. A member of a novel Arabidopsis thaliana gene family of candidate Mg2+ ion transporters complements a yeast mitochondrial group II intron-splicing mutant. Plant J. (2000) 24:489–501.[CrossRef][Web of Science][Medline]
Schreiber DN, Bantin J, Dresselhaus T. The MADS box transcription factor ZmMADS2 is required for anther and pollen maturation in maize and accumulates in apoptotic bodies during anther dehiscence. Plant Physiol. (2004) 134:1069–1079.
Shaul O. Magnesium transport and function in plants: the tip of the iceberg. BioMetals. (2002) 15:309–323.[Web of Science][Medline]
Smith RL, Banks JL, Snavely MD, Maguire ME. Sequence and topology of the CorA magnesium transport systems of Salmonella typhimurium and Escherichia coli: identification of a new class of transport protein. J. Biol. Chem. (1993) 268:14071–14080.
Smith RL, Kaczmarek MT, Kucharski LM, Maguire ME. Magnesium transport in Salmonella typhimurium: regulation of mgtA and mgtCB during invasion of epithelial and macrophage cells. Microbiology. (1998) 144:1835–1843.
Smith RL, Thompson LJ, Maguire ME. Cloning and characterization of MgtE, a putative new class of Mg2+-transporter from Bacillus firmus OF4. J. Bacteriol. (1995) 177:1233–1238.
Smyth DR, Bowman JL, Meyerowitz EM. Early flower development in Arabidopsis. Plant Cell. (1990) 2:755–768.
Snavely MD, Florer JB, Miller CG, Maguire ME. Magnesium transport in Salmonella-typhimurium magnesium-28 transport by the Cor-a Mgt-a and Mgt-B systems. J. Bacteriol. (1989) 171:4761–4766.
Szegedy MA, Maguire ME. The CorA Mg2+ transport protein of Salmonella typhimurium: mutagenesis of conserved residues in the second membrane domain. J. Biol. Chem. (1999) 274:36973–36979.
Winkler RG, Feldmann KA. PCR-based identification of T-DNA insertion mutants. Methods Mol. Biol. (1998) 82:129–136.[Medline]
Zsurka G, Gregan J, Schweyen RJ. The human mitochondrial Mrs2 protein functionally substitutes for its yeast homologue, a candidate magnesium transporter. Genomics. (2001) 72:158–168.[CrossRef][Web of Science][Medline]
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