Morphological and genetic changes induced by excess Zn in roots of Medicago truncatula A17 and a Zn accumulating mutant
© Lewis et al; licensee BioMed Central Ltd. 2012
Received: 6 July 2012
Accepted: 15 November 2012
Published: 28 November 2012
Nutrient fluxes associated with legume-rhizobia symbioses are poorly understood and little is known regarding the influence of abiotic stresses on development and maintenance of N-fixing nodules and root system architecture (RSA). We examined effects of Zn on nodule development and structure, root architecture, and expression of nodulation-related miRNAs in Medicago truncatula and the mutant, raz (requires additional Zn).
Excess Zn increased root and shoot associated Zn in both genotypes, however, raz plants had lower root associated Zn than WT plants. Roots of raz plants exposed to excess Zn had less volume, surface area, and total length compared to WT plants. Raz plants had lower lateral root number than WT plants. Excess Zn was found to increase root diameter in both genotypes. The Mn Translocation Factor (TfMn) increased in response to Zn in both genotypes; this was more pronounced in raz plants. TfZn was higher in raz plants and reduced in both genotypes in response to Zn. Nodulation was not influenced by Zn treatment or plant genotype. MicroRNA166 was upregulated under excess Zn in WT plants.
Neither the raz mutation nor Zn treatment affected nodulation, however, raz plants had altered RSA compared with WT and responded differently to Zn, implying the mutation potentially modulates RSA responses to Zn but doesn’t play a direct role in nodulation. MicroRNA166 was significantly induced in WT plants by excess Zn, warranting further investigation into the potential role it plays in controlling RSA.
KeywordsMedicago truncatula Abiotic stress MicroRNA (miRNA) Zn stress Translocation factor QRT-PCR Legume Root architecture
Medicago truncatula has been established as a model legume species because it has many desirable attributes, including, a small diploid genome, short generation time , and some level of transformability and regenerability in some genotypes . There are also a number of genetic resources available, such as, ESTs, a nearly complete sequence of gene rich regions of the genome, and genetic and physical maps . Wild-type M. truncatula plants are not known to accumulate or hyperaccumulate metals, however, M. truncatula raz (for requires additional Zn) is an ethyl methanesulfonate (EMS) generated, Zn accumulating mutant first characterized in 2003 . This mutant has been shown to accumulate greater than 10,000 μg Zn/ g d. wt. associated with root tissues and greater than 400 μg Zn/ g d. wt. associated with shoot tissues when grown in nutrient solutions containing 3 μM Zn and 2 μM Mn . When grown in nutrients solutions defined by the authors as adequate for wild-type (WT) plants, raz exhibits a high level of leaf necrosis and subsequent leaf loss, which is very similar to WT plants grown in Zn deficient conditions . Both leaf necrosis and leaf loss were partially ameliorated by providing the plants with 3 μM Zn and 0.2 μM Mn, however, raz produced less biomass than WT plants regardless of Zn treatment . Segregation analysis of progeny from crosses of WT M. truncatula and third generation raz plants revealed that a single, recessive gene is likely responsible for the raz phenotype . The mutation was localized to the upper arm of linkage group 7, as defined by Kulikova et al.  through genetic mapping studies of raz x M. truncatula A20 populations . The phenotype observed in raz plants is thought to be generated by a functional Zn deficiency . The raz phenotype, as characterized by Ellis et al. , was observed in non-nodulated plants, therefore, the phenotypic consequences of the mutation in nodulated plants have not yet been adequately explored. Zinc toxicity has been found to have numerous affects on plant growth. Studies in wheat and cucumber have revealed that high Zn decreases percent germination and inhibits root elongation as well as hypocotyl and coleoptile growth . Elevated Zn has also been found to reduce root elongation rate in 4-weeks-old seedlings of Picea abies and in rye grass . At pZn activity = 5.25 and 5.0, Ibekwe et al.  found nodulation to be inhibited in alfalfa and Zn induced root damage was thought to be the cause. Additionally, zinc toxicity was found to cause deposition of lipids on the lumen surface of xylem vessel walls and deposition of phenolic compounds on the walls of secondary vessel cells . High Zn may also inhibit cell division and elongation and increase root diameter [10, 11].
A number of studies have recently emerged covering the response of microRNAs (miRNAs) to nutrient availability [12–23]; , but to date none have reported Zn responsive miRNAs. MicroRNAs are small non-coding RNA (21 nucleotides long) that are important players in regulation of almost all aspects of plant development, and the RNA interference (RNAi) related pathways. Through RNAi machinery, genes are posttranscriptionally regulated by miRNA resulting in cleavage of mRNA, or inhibition of mRNA translation due to the binding of the miRNA to the 3' UTR (untranslated region) . RNAi pathways are important during biological development  and in adaptive plant responses to nutrient stress . Studies have revealed the role of miRNA in sulfur and phosphate homeostasis, as discussed by Chiou . One miRNA, miRNA 399, is thought to be a long distance signal in phosphate homeostasis in Arabidopsis. It has also been shown that miRNAs regulate the expression of genes responsible for Cu/Zn superoxide dismutase under Cu limiting conditions in Arabidopsis.
In shoots of A. thaliana, miR166 and the close relative miR165 have been found to be involved in regulating the class III homeodomain-leucine Zipper (HD-ZIP III) family of transcription factors . The dynamic, tissue specific regulation of these two miRNAs along with their associated targets are thought to be essential for multiple aspects of proper shoot development including, shoot apical meristem and floral development , vascular development in Arabidsopsis inflorescences , radial patterning [30, 31], and initiation of floral and lateral shoot meristems . Less is known about the function of these miRNAs in roots. Carlsbecker et al. , reported findings in support of a role for miR166b and miR165a in xylem cell identity and development in Arabidopsis. The authors propose induction of SHR (SHORT ROOT) in the metaxylem is followed by intercellular signaling which leads to the induction of SCR (SCARECROW) in the endodermis, where SHR and SCR induce miR165a/miR166b. The miRNAs are then thought to become mobile and migrate toward the metaxylem, regulating HD-ZIP III activity, leading to definition of cellular identity. Interestingly, Carlsbecker et al.  found miR166a to be undetectable in Arabidopsis roots and cite similar findings in other studies, but in M. truncatula, miR166a was found to be expressed in roots and nodules and to be involved in vascular patterning and nodule and lateral root formation . Boualem et al. , found 2x35S:MtMIR166a overexpression resulted in reduced lateral root and nodule formation accompanied by, and possibly resulting from, disorganized vascular bundling. Overexpression of MtMIR166a also led to reduced transcript levels of three of the MtHB sub-class of HD-ZIP III genes, MtCNA1, MtCNA2, and MtHB8, however, the exact function of these genes remains unknown . Expression of miR166 and the HD-ZIP III targets within the nodule are not mutually exclusive, with the miRNA and the MtHBs exemplifying co-regulation with respect to space and time. MicroRNA169 has been implicated in drought response in rice  and Arabidopsis, as well as in N and P limitation . During nodulation in M. truncatula, miR169 is essential for proper nodule development by restricting MtHAP2-1 transcripts primarily to the meristematic region of the nodule through posttranscriptional cleavage and overexpression of miR169 leads to the inhibition of nodule development presumably due to the lack of temporal-spatial control over MtHAP2-1 expression . Pant et al.  found many miR169s to be repressed during N limitation in Arabidopsis and suggested that this may be a mechanism for detecting N deficiency and at least partially initiating nodulation through the mechanisms proposed by Combier et al. . Rapid drops in miR169 transcript levels in phloem sap in response to N and P limitation also indicate the possibility of miR169 as a long-distance signal, whereby N and P deficiency are first detected in the shoots ; currently this hypothesis remains untested. As described above, miR166 and miR169 are thought to be involved in different aspects of nodulation. MicroRNA166 is likely involved in vascular bundling and it is thought that positional information derived from the stele is involved in lateral root and nodule development [38, 39]. MiR166 might also be involved later in nodule development through tight regulation of MtHB expression . The involvement of miR169 in nodulation is thought to be through maintenance of the meristematic region by restricting MtHAP2-1 expression primarily to this developmental region . The objectives of this study were to examine root architectural responses and nodule developmental processes associated with Zn in wild type Medicago truncatula or a raz mutant. We examined nodule development and structure over a 28 day time course, recorded whole root system parameters, examined metal concentrations associated with shoot and root tissues, and quantified Zn responses of the nodulation-related miRNAs, miR166 and miR169, in nodulated WT and raz plants exposed to ideal and excess Zn. The root and nodule morphological data were gathered by using a combination of confocal microscopy and WinRHIZO image analysis software. MiRNA expression levels were quantified using qRT-PCR. In attempting to develop Zn fortified legume crops, it will be very beneficial to understand the influence of Zn on root architecture and nodule formation. Studying miRNA expression in M. truncatula will give us insights into gene regulation that is applicable to many N-fixing legumes, (i.e. Soybean, Chickpea, Lentil, Common Bean), which provide essential oils and proteins with little nitrogen input compared to non-leguminous crops. Exploration of the role of miRNA in symbiotic nodule development in M. truncatula has led to the identification of many genes and their associated miRNAs essential to nodulation [34, 37, 40].
Materials and methods
Nutrient solution modified from Lullien et al.
Modified Lullien Solution
0.5x High Zn
0.5x High Zn -N
K 2 SO 4
Na 2 -EDTA
H 3 BO 3
ZnSO 4 *7H 2 O
Na 2 MoO 4
NH 4 NO 3
KH 2 PO 4
K 2 HPO 4
Fe(SO 4 )*7H 2 0
Results of GeoChem-EZ analysis of nutrients solutions in Table 1
Free Activity (−log)
1/2x High -N
RNA Isolation and qRT-PCR
Plants were harvested at two time points, 10 dpi, while nodules are developing, and at 15 dpi, when WT nodules should be mature . Upon harvesting, plants were separated into roots and shoots, massed and flash frozen in liquid N2, with any damaged or dying tissue being removed. Samples were stored in −80°C until further processing. Total RNA containing microRNA was isolated from whole root tissues of 3 bioreps (5 plants each) using the mirVana™ miRNA Isolation Kit (Ambion, Austin, TX, USA) according to the manufacturer’s instructions. Quantity and quality of total RNA was assessed using a Cary 50 UV–vis spectrophotometer (Varian Australia Pty Ltd., USA). DNase treatment was performed on 10 μg total RNA in 50 μl reactions containing 2 μl DNase using TURBO™ DNase (Ambion, Austin, TX, USA). Reverse transcription was performed on 1 μg total RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) according to manufacturer’s protocol. The same kit and RNA quantity was used for miRNA cDNA synthesis but random primers were replaced with 1 μM stem-loop primers specific to miR166 and miR169. The pulsed reverse transcription method described by Varkonyi-Gasic et al.  was used to generate cDNA for each miRNA in separate reactions. QRT-PCR was performed using 20 μl Power SYBR Green Master Mix reactions containing 2 μl of 1/10 diluted cDNA and the Applied Biosystems StepOnePlus 96-well plate system (Foster City, CA, USA) (See Additional file 2: Table S1 for primer concentrations). To amplify miR166 and miR169 along with the reference mRNA gene, actin-11, simultaneously it was necessary to modify the method established by Chen et al.  and refined by Varkonyi-Gasic et al. . In short, the 1 sec extension step at 72°C was adjusted to 10 seconds which aided in achieving adequate primer efficiencies (Additional file 2: Table S1), particularly for actin-11. Primers were obtained from IDT (Integrated DNA Technologies, USA). All miRNA real-time primers and RT primers were designed according to methods developed by Chen et al. (2005). Primer sequences for actin-11 were used from Boualem et al. . Fast PCR  was used to design and test (in silico) real-time primers. Sensitivity for primer detection was set to “3” as advised by the programmers. Amplification specificity was determined via melt-curve analysis and observing products on 4% agarose gel in TBE buffer. Mortars, pestles, and spatulas used in RNA extraction were washed and baked at 180°C overnight and RNase Zap (Ambion, Austin, TX, USA) was used to reduce the likelihood of RNase contamination. Real-time data were analyzed using GenEx Pro gene expression analysis software (version 18.104.22.168) and Systat and SAS statistical analysis software. The delta-delta Ct method was used to determine relative expression. The average relative expression of all treatments is set to zero, thus, values above zero indicate greater than average expression and values below zero indicate less than average expression. Standard error is the error of each biogroup (composed of 3 bioreps each). The Tukey’s post-hoc analysis was used to evaluate statistical differences between groups.
Confocal light microscopy
Nodule development and bacterial occupancy were monitored via confocal microscopy using SYTO13 (Molecular Probes, Inc., Eugene, OR, USA) as described by Haynes et al. . Briefly, root sections containing nodules were harvested into a Petri dish containing 80 mM PIPES buffer at 7, 14, 21, and 28 days post inoculation (dpi). A 7x-45x trinocular stereo zoom microscope (AmScope, Chino, Ca, USA) was used to visualize the nodules while they were isolated from the root segments and bisected lengthwise using a double-edged razorblade. Bisected nodule halves were then transferred to 1.5 ml microcentrifuge tubes containing 80 mM PIPES buffer. Bisects were stained at room temperature for approximately 15–20 minutes in 1 ml of 80 mM PIPES buffer containing 3 μM SYTO 13 (Molecular Probes, Inc., Eugene, OR, USA). Nodules were then rinsed thrice in 80 mM PIPES buffer to remove excess dye. Stained nodule bisects were transferred with a minimal amount of PIPES solution to appropriate slides (Corning, Inc., Corning, NY, USA). Confocal imaging was performed with a Leica TCS SP5 inverted laser scanning confocal microscope (Leica Microsystems, Exton, Pa.) at 7 dpi and with a Leica SP1 inverted confocal microscope (Leica Microsystems, Exton, Pa.) for the remaining time points. In both cases, SYTO 13 excitation was achieved with an Argon helium laser (488 nM). Images were obtained in single layers (2-D) and multiple layers (3-D) depending on nodule orientation and the desired information. ImageJ (MacBiophotonics) was used to render single maximum-intensity projections, to scale the images, and to perform general analyses. Nucleic acid distribution (and hence bacterial occupancy) is depicted as green in the confocal images. Fluorescent intensities were adjusted to minimize maximum intensities, then contrast was normalized at +20%.
Total metal analysis
At 24 dpi, raz and WT plants grown in High and Ideal Zn were harvested and separated into roots and shoots. Tissue was massed and washed in 0.001 M CaCl2 to remove weakly sorbed cations and samples stored at −20°C for future analysis. Prior to ICP-MS analysis samples were freeze-dried for 3 days and massed into 15 ml metal free polypropylene centrifuge tubes (VWR Scientific). Samples were acid digested using an open vessel microwave digestion system (CEM MARS Express, Mathews, NC). Digestions were performed in two steps, first in 750 μl of trace metal grade HNO3 at 100°C for 10 minutes and then, after samples were cooled, 250 μl of H202 was added and the samples were heated again at 100°C for 10 minutes. Digestions were then brought to 15 ml using DDI H20. Concentrations of Zn, Mn, and Mo were determined using an Agilent 7500 series ICP-MS (Santa Clara, Ca).
At 10 dpi roots of M. truncatula WT and raz grown in High and Ideal Zn were excised and examined to determine the influence of Zn on root morphology. For each genotype x treatment group, the roots of 5 plants were scanned and several root parameters, including total root length, average diameter, and total surface area, were recorded via WinRhizo Pro (Regent Instruments, Quebec, Canada). Lateral root and nodule counts were taken interactively using scanned images.
Both the raz mutation and Zn treatment have little effect on nodule phenotype and most differences observed in root morphology are genotype dependent
Nodulation may dramatically influence concentrations of root associated Zn in raz plants
qRT-PCR analysis shows mature miR166 is upregulated in response to Zn
qRT-PCR analysis shows mature miR169 is downregulated at the 15 dpi time point
Exposure of WT and raz to excess zinc resulted in many differences in root system architecture between the two genotypes. Interestingly, differences in lateral root number were evident between genotypes but no difference was noticed in nodule number or development; implying that while the raz mutation does inhibit lateral root formation it does not inhibit the closely related process of nodulation. Ibekwe et al.  found high Zn activities delayed or inhibited nodulation in Medicago sativa inoculated with S. meliloti, but did not inhibit N-fixation in developed nodules. Conversely, Zn deficiency has been shown to disturb symbiotic N-fixation by altering other processes within the host plant, such as nutrient transport and assimilation . Expression of a Krüppel-like zinc finger protein is integral to the formation of the N-fixation zone in M. truncatula. In these ways, Zn plays a significant role in nodule development. In our study, the process of nodulation was unaffected by the Zn treatment in both genotypes.
Ellis et al.  found Zn tissue concentrations in non-nodulated M. truncatula WT and raz to be dependent upon Mn concentrations and vice versa, with raz exhibiting greater sensitivity to variations in concentration of either nutrient. For instance, at 3 μM Zn and 2 μM Mn, raz plants had root Zn levels >10,000 μg g-1 d. wt. compared with raz at 3 μM Zn and 0.2 μM Mn which had <4,000 μg g-1 d. wt. In contrast, WT plants showed little difference in root Zn levels in either of these treatments. Also, raz had greater root Mn levels at 3 μM Zn and 2 μM Mn (~3,000 μg g-1 d. wt.) and 0 μM Zn and 2 μM Mn (>4,000 μg g-1 d. wt.) compared with 1 μM Zn and 2 μM Mn (<2,000 μg g-1 d. wt. ). Under these and several other Zn:Mn ratios raz plants had greater root Zn and Mn concentrations than WT plants. Using the same nutrient solution with Zn and Mn concentrations (3 μM Zn and 0.2 μM Mn and pH 5.5) that Ellis et al.  showed to be ideal for raz in non-nodulated treatments, we made several unsuccessful attempts to nodulate WT and raz plants. To evaluate why the plants weren’t nodulating, we used GeoChem EZ to model the nutrient solutions used by Ellis et al.  and found the pZn activity to be around 5.7 at pH 5.5. Ibekwe et al.  showed that yellowing and necrosis in young leaves of M. sativa occurred at a pZn activity = 8, 5.25, and 5.0, with plant health improving after 10 days of treatment at pZn activity = 8. However, at pZn activity = 5.25 and 5.0 they found overall plant growth was stunted and no nodulation was achieved. Lack of nodulation was attributed to damaged root tissue. Ibekwe et al.  were examining the interaction of Cd/ Zn toxicity on M. sativa and S. meliloti, but the Zn studies were carried out at a Cd activity that was found to not influence the growth or nodulation of the plants or rhizobia. There is little research on the influence of metal concentrations on nodulation in M. truncatula so it is possible that the response to heavy metals may be slightly different than that of M. sativa. Regardless, we have shown some agreement with the influence of Zn on nodulation between the species.
Nodulation appears to alter the relationship between Zn and Mn uptake and allocation in M. truncatula compared to non-nodulated experiments shown in previous studies [3, 48]. This relationship seems to be altered more dramatically in the raz mutant, where a greater proportion of Zn was allocated to the shoots in both High and Ideal Zn compared with WT and where a greater proportion of Mn remained associated with the roots in Ideal Zn (Figure 4xa, b). In our study, WT and raz plants had significantly higher levels of Zn in root and shoot tissues in the High Zn treatment compared to Ideal conditions (Figure 3a, b). Ellis et al. (2003) reported greater Zn concentration in roots verses shoots and elevated Zn conditions are known to influence metal distribution in the plant by inhibiting translocation of Zn to the shoots leading to Zn accumulation in the roots (Rout and Das 2003). Interestingly, WT plants had significantly more Zn associated with root tissues compared to raz under High Zn conditions. This finding is contrary to the results of Ellis et al. (2003) and Lopez-Millan et al. (2005) in non-nodulated plants, suggesting nodulation affects Zn uptake and distribution in raz plants. Since the pZn activities in nutrient solutions used in our studies are different than those used by Ellis et al. (2003) and Lopez-Millan et al. (2005) it is difficult to compare the actual tissue concentrations, though our results for root and shoot Zn concentrations in WT plants do fall roughly in the range that one may expect based on their results. Perhaps the lower levels of Zn in the roots is an artifact of the excess Zn being shuttled to the shoots (TfZn is higher in raz). It could also be related to the smaller size of raz roots given that surface area and volume are lower in this genotype. We also observed greater uptake of Mn by raz roots exposed to Ideal Zn versus High Zn (Figure 3c). Ellis et al. (2003) and Lopez-Millan et al. (2005) found raz plants had greater root tissue associated Zn and Mn levels than WT plants under various Zn-Mn regimes and that raz plants tend to take up more Mn with increasing Zn concentrations, with the exception of [Zn] = 0. These results again point to a possible influence of nodulation on the partitioning of metals within the plant and warrants further experimentation to deduce the mechanisms underlying this dramatic, possibly nodulation induced, change in Mn (and Zn) uptake by raz mutants.
There is much to learn concerning the nutrient demands generated by nodulation or the fluxes in nutrient uptake and allocation possibly associated with the formation and maintenance of nodules, as well as symbiosis as a whole. The potential nodulation-related changes we observed in Zn and Mn uptake in M. truncatula may be indicative of nutritional adjustments necessary to properly form and maintain the symbiosis. Previous studies in non-nodulated plants have found no correlation between MnSOD and total Mn associated with plant tissues of either genotype, however, a strong correlation was found between tissue associated Zn and ZnSOD . The authors found ZnSOD levels were lower in raz plants exposed to high Zn compared with WT in the same treatment and compared with both genotypes in lower Zn, indicating that Zn is less available in raz roots past a certain threshold. Concentrations of root associated Mo were increased in response to Zn in WT but not raz. This may be indicative of differences in the plant genotypic responses to excess Zn. If raz plants are less sensitive to high levels of Zn, then the Zn tolerance mechanisms, such as the release of metal sequestering compounds into the rhizosphere, would be induced to a lesser extent compared with WT, thereby leading to lower levels of root associated Mo. However, since previous researchers have found no such association in other species and no work has been done regarding this type of interaction in Medicago species, this warrants further study.
As discussed earlier, excess Zn is known to affect several developmental parameters (i.e. increased root diameter, inhibition of root elongation, and inhibition of hypocotyl and coleoptile growth). Given that miR166 is thought to play roles in root cell identity , our findings, even though we are unable to determine which miR166 family member(s) is/are the source of the mature miRNA, may allude to part of the genetic mechanism by which Zn inhibits cell division. It is not uncommon for researchers to report increases in root diameter along with decreases in root elongation [56–59]. It is also thought that decreases in root elongation can potentially arise from decreases in cell division , understandably, given the intimate connection between the two processes . Boualem et al. (2008) showed that MtMIR166a is involved in nodule development as well as root architecture in nodulated and non-nodulated M. truncatula. They showed a progressive trend in downregulation of MtMIR166a in nodules of M. truncatula moving from 1, 3, 8, and 21 dpi, with the 21 dpi point showing significant downregulation. Here, we are reporting two time points within this range and have found a similar trend in mature miR166 expression; downregulation from whole nodulated roots in Ideal Zn. Boualem et al. , also showed that overexpression of miR166 in M. truncatula via 2x35S promotion leads to a decrease in nodule number and lateral root formation accompanied by dramatic reordering of vascular bundling patterns. We did not investigate vascular bundling patterns, but we found no experimental effect on nodule number and no treatment effect on lateral root number (Figure 2a). We did note a genotypic difference in lateral root number, where WT plants generated significantly more lateral roots than raz plants (Figure 2a); the same phenomenon was observed in lateral root density (Additional file 3: Figure S2). Boualem et al.  examined the effects of overexpressing miR166 through strong promotion and our study was constructed to analyze the behavior of miR166 in response to Zn in non-transgenic plants. The upregulation we witnessed is likely inadequate to reproduce the drastic changes in phenotype observed by Boualem et al.  and is potentially indicative of other, more subtle, roles of miR166 in Zn response. The suggested role of miR166 directed regulation of HD-ZIP III transcription factors in vascular bundling and patterning insinuates Zn induction of miR166 is possibly related to changes in root vascular structure necessary to withstand, or induced by, the elevated Zn condition.
Investigation into the functions of miRNAs in plants is rapidly evolving, revealing their roles in a multitude of biological processes from basic development to abiotic stress response via long distance phloem transport. Here we have shown that miR166 is upregulated in response to chronic Zn exposure in nodulated M. truncatula WT and raz. The effect of Zn on miR166 is statistically significant at the 15 dpi sample point. Given that Zn exposure did not generate phenotypes similar to those with 2x35S:MtMIR166a overexpression , our results may imply a previously unidentified role for miR166 in Zn response. WT plants developed significantly more lateral roots than raz plants while miR166 expression was very similar across genotypes. Differences in lateral root formation were not associated with Zn treatment, yet upregulation of miR166 was. We found no relationship between Zn exposure and expression of miR169. We also found no difference in expression of miR169 between genotypes. Complimentary to other experiments monitoring expression of the precursor, MtMIR169a, we found mature miR169 to be downregulated at the 15 dpi point compared to the 10 dpi point . Suggesting that expression of mature miR169 in whole nodule containing root tissues may mimic what has previously been observed in root nodules. However, we observed no difference in nodule formation in response to Zn or between genotypes and given miR169 expression is related to nodule development and function by limiting the expression of MtHAP2-1to the meristematic region  it is unlikely that examination of nodule tissue only would show any meaningful trends.
In summary, nodule development appears to be unaffected by the raz mutation, suggesting the alteration in Zn partitioning induced by the mutation is of little consequence concerning this process. Excess Zn also appears to have little influence on nodule development or number in M. truncatula, as has been observed in M. sativa. The major root architectural Zn responses observed in this study were amongst genotypes, indicating the raz mutation modifies root development in relation to WT plants and that this modification alters phenotypic responses to Zn. We have provided evidence that nodulation may alter the relationship between Zn and Mn concentrations associated with root and shoot tissues and possibly hinder the Zn accumulating abilities previously characterized in raz. MiR166 is induced in roots of M. truncatula WT and raz in response to Zn and this appears to be maintained through time under chronic Zn exposure. Given the proposed mechanism by which miR166 is thought to regulate, at least partially, root cell identity; our results may imply some part of the mechanism by which excess Zn inhibits cell division. Further studies should investigate the role that miR166 plays in Zn responses to determine the precise function of this miRNA in chronic Zn exposure. The findings we report here provide insight into how legume roots respond to Zn and add to previous research which also found moderately high Zn levels to have little effect on nodule development, provided Zn concentrations were beneath the threshold where nodulation is drastically inhibited. Future research should focus on the nutritional demands that nodule development places upon legumes and how this may alter nutrient fluxes within the plant. Investigation into the roles of miRNA in Zn stress may also prove fruitful in attempts to further understand genetic mechanisms governing Zn induced phenotypic responses.
Requires additional zinc
Root system architecture
We appreciate seed gifts from Michael Grusak, Hongyan Zhu, and Troy Bass. Sinorhizobium meliloti stock was provided by Janine Sherrier, for which we are very grateful. We thank Olga Tsyusko, Mohammad Babar Ali, Xiaoyun Jia, Ligang Ren and B. Usha for advice on qRT-PCR and general RNA/miRNA methodology, and Jason Unrine for metals analysis via ICP-MS. We also thank Maria Labreveux and Xia Yu for assistance with statistical analysis, and Joseph V. Kupper and Rebecca Sims for assistance with general lab work.
This work was funded by the University of Kentucky Department of Plant and Soil Science.
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