JXB Advance Access originally published online on March 2, 2008
Journal of Experimental Botany 2008 59(3):697-704; doi:10.1093/jxb/erm351
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© 2008 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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RESEARCH PAPER |
Laser microdissection-assisted analysis of the functional fate of iron deficiency-induced root hairs in cucumber
1Dipartimento di Scienze Agrarie e Ambientali, University of Udine, Via delle Scienze 208, I-33100 Udine, Italy
2Institute of Plant and Microbial Biology, Academia Sinica, 115 Taipei, Taiwan
* To whom correspondence should be addressed. E-mail: wosh{at}gatesinica.edu.tw
Received 9 November 2007; Revised 12 December 2007 Accepted 13 December 2007
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Abstract |
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Iron ranks fourth in the sequence of abundance of the elements in the Earth's crust, but its low bio-availability often limits plant growth. When present in suboptimal amounts, the acquisition of iron by plants is aided by a suite of responses, comprising molecular and developmental changes that facilitate the uptake of iron from sparingly soluble pools. The expression of genes involved in the mobilization of iron (CsHA1), the reduction of ferric chelates (CsFRO1), and in the uptake of ferrous iron (CsIRT1) was investigated in epidermal cells of Fe-sufficient and Fe-deficient cucumber (Cucumis sativum L.) roots using the Laser Microdissection and Pressure Catapulting (LMPC) method. Growing plants hydroponically in media deprived of iron induced the differentiation of almost all epidermal cells into root hairs. No root hairs were formed under iron-replete conditions. The formation of root hairs in response to Fe starvation was associated with a dramatic increase in message levels of CsFRO1, CsIRT1, and the iron-inducible H+-ATPase isoform CsHA1, when compared to epidermal cells of Fe-sufficient plants. On the contrary, transcripts of a housekeeping ATPase isoform, CsHA2, were not detected in root hairs, suggesting that Fe-deficiency-induced acidification is predominantly mediated by CsHA1. These data show that the formation of root hairs in response to iron deficiency is associated with cell-specific accumulation of transcripts that are involved in iron acquisition. The results also show that this includes the differential regulation of ATPase isoforms with similar function, but supposedly different characteristics, to counteract the imbalance in nutrient supply efficiently.
Key words: ATPase, cucumber, iron uptake, laser microdissection, root development, root hairs, roots
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Introduction |
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Despite its usually high abundance in soils, iron is generally present at low solubility, causing yield losses in more than two-thirds of the world's arable soils. Since plants are the main source of iron in most diets, iron deficiency is a major nutritional disorder that affects two billion people worldwide (http://www.who.int/en). According to their mechanisms to acquire iron from soils, plants have been classified into two groups, referred to as strategy I and strategy II (Römheld, 1987; see Mori, 1999; Curie and Briat, 2003, and Schmidt, 2003, for reviews). Similar to micro-organisms, grasses produce and release iron-avid (phyto)siderophores (PS) and take up the ferric iron-loaded PS–metal complex by an iron-inducible, membrane-bound transporter (strategy II). Phytosiderophores are produced by transamination of nicotianamine (NA) via NA aminotransferase (NAAT), catalysing the amino group transfer of NA to form PS. Strategy I plants, comprising all plants except the Graminaceae, lack the capability to synthesize PS. Acquisition of iron in this group relies on a reduction step of ferric iron bound to chelates and subsequent uptake of the liberated ferrous iron. Reduction of ferric chelates is mediated by an iron-regulated oxido-reductase (Ferric Reductase Oxidase, FRO) expressed in root epidermal cells (Robinson et al., 1999). The reduced iron is transported across the plasma membrane by IRT1 (Iron Regulated Transporter1), a transporter belonging to the ZIP (Zn-Fe-regulated transporter) family of metal transporters first identified in Arabidopsis (Eide et al., 1996). Homologues of IRT1 have been cloned from tomato (Eckhardt et al., 2001), pea (Cohen et al., 2004), and recently from cucumber (Waters et al., 2007). An IRT1 homologue was also identified in rice (Bughio et al., 2002), suggesting that an alternative, conserved route for the entry of iron into the cell exists in strategy II plants. In Arabidopsis, FRO2 and IRT1 are strictly co-regulated by both a local signal perceived by the roots and a systemic signal conveying the shoot's iron status by an as yet unidentified mobile signal (Vert et al., 2003). A root-specific bHLH protein controlling both genes was recently identified in tomato and Arabidopsis (Ling et al., 2002; Colangelo and Guerinot, 2004; Jakoby et al., 2004; Yuan et al., 2005), and has been named FER in tomato and FIT1 (Fe-Induced Transcription Factor 1) in Arabidopsis (Bauer et al., 2007). Both AtFRO2 and AtIRT1 are also regulated post-transcriptionally, probably as a ‘rescue’ system to avoid the generation of hydroxyl radicals by excess iron (Connolly et al., 2002, 2003).
The solubility of iron strongly depends on proton activity; it has been estimated that with every unit of pH increase the iron solubility decreases by 1000-fold. Rhizosphere acidification is of the utmost importance for the acquisition of iron in plants relying on the strategy I-type response; the PS-based iron uptake is less sensitive to high pH and iron-inducible rhizosphere acidification has not been reported for strategy II species. Lowering the pH in response to iron deficiency is mediated by P-type H+-ATPases, catalysing the electrogenic transport of protons from the cytosol across the plasma membrane (Dell'Orto et al., 2000; Schmidt et al., 2003). An iron-responsive H+-ATPase gene, CsHA1, has recently been isolated from cucumber roots (Santi et al., 2005). The regulation of CsHA1 differs from that of the housekeeping isoform CsHA2, the expression of which was found to be unaffected by the iron status of the plants. Both H+-ATPases show high similarity at the protein level, suggesting differences in expression pattern among the isoforms being the decisive factor(s) for the preferential expression of either gene. Despite the potential importance of proton extrusion in the iron acquisition of strategy I plants, the capacity for acidifying the rhizosphere largely varies among species. Interestingly, this variation appears to be associated with iron deficiency-induced developmental changes in the rhizodermis (Römheld and Kramer, 1983).
The physiological responses to Fe-deficiency are localized in discrete zones of the root and are mainly confined to the outermost cell layers. For example, in red pepper, the ability of extruding protons is restricted to the sub-apical region of the root and to root hairs, where, simultaneously, ferric reduction activity and organic acid accumulation have been observed (Landsberg, 1986). In Arabidopsis, transcripts of FRO2 and IRT1 are preferentially localized in the outer cell layer of Fe-deficient roots (Connolly et al., 2003; Vert et al., 2002). In cucumber, H+-ATPase was immuno-detected in epidermal, endodermal, cortical, and root hair cells (Dell'Orto et al., 2002). Similarly, in tomato, H+-ATPase protein accumulation was shown in transfer cells formed in the rhizodermis of Fe-deficient plants (Schmidt et al., 2003). The localized accumulation of transcripts and their products of genes involved in iron acquisition implies, that the expression of these genes is controlled by cell-specific promoters.
The induction of the physiological Fe acquisition processes is paralleled by changes in post-embryonic development, such as reduction in primary root growth, alterations in the root architecture, and in the structure and ultrastructure of epidermal cells. In particular, the formation of extra root hairs is a ubiquitous response to Fe deficiency and has been reported for several strategy I species such as sunflower (Landsberg, 1996), tomato (Schikora and Schmidt, 2002; Graziano and Lamattina, 2007), and Arabidopsis (Schikora and Schmidt, 2001). Root hairs are long tubular extensions from rhizodermal trichoblasts controlled by multiple gene loci (Grierson and Schiefelbein, 2002; Larkin et al., 2003). The formation of root hairs is affected by the environment and responsive to the availability of mineral nutrients (Ma et al., 2001; Schmidt and Linke, 2007; Schmidt, 2008). A higher root hair density or an increase in root hair length greatly enlarge the surface area of the root and allows for a greater soil volume to be explored. This is of particular importance for the uptake of essential nutrients with limited mobility in the soil solution such as phosphate and iron.
Information about a co-ordinate expression of the genes involved in Fe acquisition in single cell types is lacking. The aim of the present work was to clarify the relationship between form and function of cucumber root epidermal cells in the response to Fe-deficiency. Functional analysis of root hairs was permitted by the isolation of epidermal cells from paraffin-embedded root sections of hydroponically grown plants by Laser Microdissection (LM). This technique increases the resolution of gene expression to the cell-specific level, which is critical to elucidate responses that are restricted to a particular tissue or cell type. Nanogram quantities of total RNA extracted from laser-microdissected tissues were linearly amplified by T7 RNA polymerase and used in gene expression analyses (see Kerk et al., 2003; Nelson et al., 2006; Day et al., 2007, for reviews). It is shown that the major players in the acquisition of iron such as those responsible for mediating the solubilization of iron (CsHA1), the reduction of ferric chelates (CsFRO1), and the uptake of iron (CsIRT1), were co-expressed in root epidermal cells. Growing the plants in iron-deficient media induced the differentiation of rhizodermal cells into root hairs. Iron deficiency-induced changes in gene expression further included a decrease in the expression of the housekeeping H+-ATPase isoform CsHA2 in root hairs, indicating that rhizosphere acidification by iron-deficient cucumber roots is predominantly mediated by the inducible isoform CsHA1.
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Materials and methods |
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Plant material and growth conditions
Four-day-old cucumber seedlings (Cucumis sativus L., cv. Lungo della Cina) were hydroponically grown for 7 d in black plastic pots (4 plants per pot) containing 2.0 l of continuously aerated nutrient solution either without (–Fe plants) or with 40 µM Fe(III)-EDTA (+Fe plants). The nutrient solution was renewed every 2–3 d and had the following composition: 2 mM Ca(NO3)2, 0.7 mM K2SO4, 0.5 mM MgSO4, 0.1 mM KCl, 0.1 mM KH2PO4; 10 µM H3BO3, 0.5 µM MnSO4, 0.5 µM ZnSO4, 0.2 µM CuSO4, 0.01 µM (NH4)6Mo7O24. The pH was adjusted to 6.0 with 1 N NaOH. Plants were grown under a photon flux density of 150 µmol m–2 s–1 (16 h photoperiod), at a 25/20 °C (day/night) temperature regime with a relative humidity between 50% and 70%.
Microscopical analysis of cucumber roots
Roots of two plants were collected and serial hand-cut cross-sections of roots from +Fe and –Fe plants were analysed with an Imager Z1 (Zeiss, Jena, Germany) microscope by bright-field microscopy. For cell wall visualization, sections were examined with a Axiophot fluorescence microscope (Zeiss, Germany) using a G365 UV excitation filter, an FT 395 chromatic beam splitter and an LPH 20 barrier filter.
Visualization of proton extrusion
Spatial localization of acidification capacity was determined by embedding the roots of intact plants in an agar (0.7%) medium containing 0.5 mM CaSO4 and the pH indicator Bromocresol Purple (0.005%) for 15 min. The pH of the medium was adjusted to pH 6.0. The roots had been grown for 7 d in either full nutrient solution or Fe-free medium. Rhizosphere acidification was recorded with a motorized SteREO Discovery.V12 (Zeiss, Jena, Germany) steromicroscope combined with an AxioCam digital microscope camera and AxioVision image analysis software (Zeiss, Jena, Germany).
Laser Microdissection Pressure Catapulting (LMPC)
Root tips from three plants were cut 5–10 mm from the apex and fixed for 4 h in 3:1 v:v ethanol:acetic acid on ice. Control and Fe-deficient plants were separately collected. Vacuum was applied three times for a few seconds and rapidly released. Fixed roots were dehydrated in glass vials at room temperature in a graded ethanol series [40 min, 70% (v/v) ethanol; 30 min, 90% (v/v) ethanol; 30 min, 100% (v/v) ethanol; 30 min, 100% (v/v) ethanol, 30 min, 100% (v/v) xylene; 30 min, 100% (v/v) xylene]. Specimens were subsequently transferred to plastic cassettes with pads and infiltrated with Paraplast (Mc Cormick Scientific, St Louis, MO, USA) at 59 °C. Paraplast was replaced three times at intervals of 30 min. Fixed roots were embedded in Paraplast using 1 cm metal base moulds. Blocks were first cooled down to room temperature and then placed at 4 °C for easy unmoulding. The blocks were kept in plastic bags at 4 °C. Eight µm-thick slices were sectioned on a rotary microtome (Leica, Bensheim, Germany). Sections were floated in DEPC-water at 42 °C on PEN-covered glass slides (P.A.L.M. Microlaser Technologies GmbH, Carl Zeiss MicroImaging GmbH, Germany), and dried at 42 °C for 1 h. Immediately prior to LMPC, the sections were de-paraffinized twice for 2 min each in 100% (v/v) xylene and then air-dried. The de-paraffinized sections were microdissected with a P.A.L.M. Laser-Microbeam System (Carl Zeiss MicroImaging GmbH, Germany). Epidermal cells in the primary root zone were dissected from at least 12 sections of control or Fe-deficient roots (600–1000 cells), and separately placed in 0.5 ml sample tubes with adhesive lids.
RNA extraction and cDNA synthesis
Total RNA was isolated from apical segments (about 5–10 mm from the tip) of primary and lateral roots of a minimum of three +Fe or –Fe plants with the RNeasy Plant Mini Kit (Qiagen) according to the manufacturer's instructions. Nucleic acid quantity was evaluated by using a NanoDrop ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies, Wilmington, USA). About 1 µg of total DNase-treated RNA (Turbo DNase, Ambion) was reverse-transcribed using an oligo-dT primer and the Superscript® III Reverse Transcriptase (Invitrogen Co, Carlsbad, CA) in a total volume of 20 µl.
RNA extraction and RNA amplification from LMPC-captured cells
RNA from cells was extracted using the Absolutely RNA Nanoprep Kit (Stratagene, La Jolla, CA, USA) with minor changes with respect to the manufacturer's instructions. Briefly, 30 µl of TRIzol Reagent (Invitrogen) was applied to the special adherent lid containing laser captured cells of the microcentrifuge tube. After inverting and vortexing the tube, 70 µl of lysis buffer was added and mixed with an equal volume of 70% ethanol. This mixture was transferred to the RNA-binding column, and DNase treatment and washing were performed subsequently. RNA was eluted in 12 µl of elution buffer warmed to 60 °C. RNA amplification was performed using the RiboAmp RNA Amplification kit (Arcturus Molecular Devices, Mountain View, CA, USA) according to the manufacturer's instructions. Briefly, first-round cDNA synthesis was performed with an oligo(dT) primer with the T7 promoter. One µl of T7 oligo(dT) primer was added to 10 µl of RNA and heated to 65 °C for 5 min. After cooling to 4 °C, 9 µl of the complete first strand synthesis mix was added. After a 45 min incubation period at 42 °C and subsequent cooling to 4 °C, 2 µl of nuclease mix was added and the reaction was carried out for 20 min at 37 °C. The quality of input RNA was assessed after first strand synthesis using real-time RT-PCR. Five µl of 1:10 diluted cDNA were used in the PCR reaction. Second-strand synthesis was started by the addition of 1 µl of exogenous primers and denaturation at 95 °C for 2 min, followed by chilling at 4 °C and addition of 30 µl of second strand mix. The reaction was performed at 37 °C for 10 min. The cDNA was purified according to the manufacturer's instructions and eluted with 16 µl of DNA elution buffer. For in vitro transcription, 16 µl of double-stranded cDNA was used in a 40 µl total volume reaction containing the T7 RNA polymerase, incubated at 42 °C for 4 h, and then digested with DNase. The aRNA obtained by in vitro transcription was purified according to the manufacturer's instructions and eluted with 30 µl of RNA elution buffer.
Real-time RT-PCR
For the expression analysis in root apical segments by real-time RT-PCR, the cDNA synthesis reaction mixture was diluted and about 25 ng of the initial RNA were used for PCR. Real-time RT-PCR was performed using the Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen) in a 20 µl total volume. A DNA Engine Opticon 2 (MJ Research Inc., Waltham, MA, USA) system was used, imposing the following standard thermal profile: 2 min at 50 °C, 5 min a 95 °C, followed by 40 cycles of 20 s at 95 °C, 30 s at 56 °C, 30 s at 71 °C. In addition, a melting curve analysis was performed which resulted in single product-specific melting temperatures. Gene-specific primer couples had been previously evaluated using the BLASTN algorithm (Altschul et al., 1997). To enable detection of contaminating genomic DNA, PCR was performed with RNA as template. A mean normalized expression for each target gene was calculated by considering a 100% efficiency and then normalizing to the level of ubiquitin, imposing transcript abundance of ubiquitin = 100 arbitrary units. Mean normalized gene expression values were graphed assigning a value of zero to no expression. Three technical and two biological replicates were performed.
For gene expression analysis in LMPC-collected cells, 8 µl of aRNA was reverse-transcribed using random hexamers and the Superscript® III Reverse Transcriptase (Invitrogen) in a total volume of 20 µl. Real-time RT-PCR was performed using the same conditions described above, using 10-fold diluted cDNA. Three technical replicates for each of the two biological replicates were performed.
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Results |
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Morphological root responses to Fe deficiency
As part of the key responses of strategy I plants to Fe deficiency, cucumber roots increase their ability to acidify the rhizosphere by the extrusion of protons (Fig. 1). The active zone is restricted to the swollen sub-apical region of the root, which corresponds to the root hair zone. Serial cross-sections in this zone revealed that almost all epidermal cells developed into root hairs, both in the primary root (Fig. 2A, B) and in laterals (Fig. 2C). At about the same distance from the tip, roots from Fe-sufficient plants showed an earlier developmental stage without root hairs, as shown by the presence of lateral root cap cells (Fig. 2D). The differences were due to an Fe deficiency-induced growth restriction of the primary root, a common response to Fe starvation (Schmidt, 1999). Roots of Fe-sufficient plants did not form hairs at later developmental stages (Fig. 2D). In order to avoid biased results due to differently aged zones in control and Fe-deficient roots during LM experiments, only sections showing metaxylem formation were used for subsequent experiments. Comparable cross-sections from the primary zone of control and Fe-deficient root are shown in Fig. 3. The autofluorescence caused by suberin and lignin deposits showed protoxylem and developing metaxylem, the outer walls of epidermal cells, and traces of the radial walls of endodermal cells. Epidermal cells appeared partly collapsed, probably together with the outer cortical cells.
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Gene expression analysis in Fe-deficient roots
The expression of the ferric reductase gene CsFRO1 (accession no. AY590765; Waters et al., 2007), the iron transporter CsIRT1 (accession no. AY590764; Waters et al., 2007), and two H+-ATPase genes, CsHA1 and CsHA2 (accession nos AJ703810 and AJ703811; Santi et al., 2005), in apical root segments was investigated by real-time RT-PCR. Expression analysis was focused on discrete regions of cucumber roots; 5–10 mm from the apex of the primary root and of lateral roots were collected for RNA extraction. Since Fe deficiency induces the formation of very short lateral roots, laterals of Fe-deficient plants were cut off at the axis of the primary root. The expression of the iron acquisition genes was normalized against ubiquitin, which was found to be unaffected by Fe deficiency (data not shown).
CsFRO1 and CsIRT1 transcripts were not detected in control roots, but Fe deficiency strongly increased their steady-state levels (Fig. 4A). Similarly, CsHA1 transcript abundance was very low in control roots and was strongly increased (80-fold) in Fe-deficient roots. CsHA2 transcripts were detected both in control and treated roots and appeared to be unaffected by the iron nutritional status. In control roots, CsHA1 transcripts represented only about 4% of the total HA (CsHA1 and CsHA2) mRNAs, whereas in roots of Fe-deficient plants the relative level of CsHA1 messengers was increased up to 70%. In root apical sections the expression of CsHA1 was markedly higher when compared to what we have previously observed for whole roots of Fe-deficient plants (Santi et al., 2005).
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Gene expression analysis in LMPC-derived epidermal cells
The Laser Microdissection Pressure Catapulting (LMPC) technique was applied to cucumber rhizodermal cells to allow for a single cell type-specific expression analysis of the iron-responsive genes. Root hair cells and non-hair epidermal cells were separately collected from Fe-deficient and control roots, respectively (Fig. 5). Cells were captured from sections showing metaxylem formation. In order to avoid a stochastic behaviour for low abundance messages, a minimum of 600–700 cells was collected from at least 12 sections for each treatment. LMPC was repeated in two biologically distinct experiments.
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Gene expression was analysed by real-time RT-PCR in LMPC-isolated root hairs from Fe-deficient roots and compared to epidermal cells collected in the same root zone from Fe-sufficient roots. RNA was purified from the collected material and amplified by one round of T7 RNA polymerase-based RNA amplification, yielding an average amount of 502±87 ng. The linearity of amplification was evaluated using real-time RT-PCR by comparing the expression level of transcripts before and after RNA amplification (data not shown). The comparison confirmed the manufacturer's quality test. The quality of RNA from laser-captured cells was checked by using different pairs of primers for the same gene, each differing in the distance from the 3' end of its corresponding sequence. In this way, it was estimated that the average size of the one-round-amplified RNA from laser-captured cells was around 400 bp. Expression of the ubiquitin gene was shown to be non cell-specific (Kerk et al., 2003; Inada and Wildermuth, 2005) and was used as an internal control.
Expression analysis of CsFRO1, CsIRT1, and the two CsHA genes in epidermal cells of cucumber roots is shown in Fig. 4B. CsFRO1 and CsIRT1 transcripts were not detected in epidermal cells of Fe-sufficient plants. On the contrary, high message levels were observed in root hairs of Fe-deficient plants, suggesting preferential expression of these genes in this specific cell type. Moreover, the expression level of these genes was almost one order of magnitude higher (in the case of CsFRO almost two orders of magnitude) when compared with that in root apical segments. CsHA1 expression was low in epidermal cells of control plants, but 35-fold up-regulated in root hairs (Fig. 4B). These results confirmed the major roles of these genes in the Fe acquisition of cucumber (Santi et al., 2005; Waters et al., 2007). Moreover, the housekeeping H+-ATPase isoform CsHA2 showed high message levels in control epidermal cells, whereas no transcript was detectable in root hairs. The comparison made was between root hairs formed during Fe-deficiency and rhizodermal cells from Fe-sufficient plants. This, of course, does not mean that those genes are not expressed in cells or tissue types other than in the rhizodermis. Nevertheless, under control conditions, CsFRO and CsIRT are neither expressed in the epidermis nor in other tissues of the sub-apical zone of the roots, suggesting that those genes are most probably predominantly up-regulated in root hairs.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In this study, the cell type-specific expression of genes involved in the acquisition of iron in root epidermal cells of cucumber grown in hydroponic culture was investigated. Root hair cells formed in response to Fe deficiency were captured by LM and compared with epidermal (non-hair) cells from roots grown under control (Fe-sufficient) conditions. The results obtained with LM-captured cells were validated by analysing the expression pattern of the genes under investigation in apical root segments in parallel. The single cell type-specific approach eliminates the transcriptional noise due to the mixture of different tissues and avoids the dilution of transcripts with low abundance. In addition, the transcriptional regulation of different stress-induced genes can be compared within the cell type in which they are preferentially expressed, which may gain insights into the potential roles in, and the relative importance of particular components for, a given stress response. In the present study, this was most evident in the case of the housekeeping H+-ATPase isoform CsHA2, which was expressed in rhizodermal cells of Fe-sufficient plants, but was not detectable in Fe-deficiency-induced root hairs. This argues for the paramount importance of CsHA1 in the iron stress response, which appears specifically to mediate the Fe-deficiency-induced acidification. The increase in CsHA1 message level is probably mirrored by a protein accumulation in root hairs. A polarized expression pattern of the H+-ATPase protein, preferentially on membranes lining the outer periclinal walls, has been observed in Fe-deficient rhizodermal cells of tomato (Schmidt et al., 2003), and an accumulation of the protein at the whole root level was immunodetected in Fe-deficient cucumber (Santi et al., 2005).
Although considered as being part of the core of the reduction-based iron acquisition strategy, both iron stress-induced root hair formation and rhizosphere acidification are comparatively weak responses in Arabidopsis. Only relatively few epidermal cells were found to be reprogrammed to root hair cells in response to Fe deficiency (Schikora and Schmidt, 2001). An increase in the absorptive surface is achieved by the formation of bifurcated root hairs (Müller and Schmidt, 2004). The iron deficiency-inducible H+-ATPase isoform CsHA1 shows 88% identity to the Arabidopsis ATPase AHA2, which is only moderately up-regulated in response to iron starvation in the Col-0 accession (S Santi and W Schmidt, unpublished data). On the contrary, both responses are very prominent in cucumber roots. Almost all epidermal cells were found to differentiate into root hairs under Fe-deficient conditions, which was associated with a robust acidification of the rhizosphere (this study; Dell'Orto et al., 2002; Santi et al., 2005; Waters et al., 2007). No marked differences between Arabidopsis and cucumber were observed with regard to the gene expression level of the ferric reductase and the transporter of ferrous iron (Vert et al., 2003; Waters et al., 2007). The obvious differences in root hair formation and acidification capacity between the two species make it tempting to speculate that both responses are functionally linked. A co-regulation of root hair formation and proton efflux is advantageous in ecological terms; both responses are crucial to access low mobility iron pools. More work is necessary to verify this assumption. In any case, the results presented here argue that functional and developmental changes in response to environmental stimuli occur in close association.
The co-expression observed for CsHA1, CsFRO, and CsIRT may be controlled by a common trans-acting regulatory factor. In Arabidopsis, a suite of 72 iron-responsive genes, among them AtFRO2 and AtIRT1, are regulated by the bHLH transcription factor FIT1 (Colangelo and Guerinot, 2004; Jakoby et al., 2004; Yuan et al., 2005). Consistent with such a function attributed to this gene, FIT1 promotor activity was detected in the outer cell layers of the differentiation zone and was found to be induced by Fe deficiency (Colangelo and Guerinot, 2004; Jakoby et al., 2004). Interestingly, AtAHA2 is not FIT1-regulated, suggesting a more complex control of the strategy I syndrome. Ethylene was shown to be involved in root hair formation in Arabidopsis (Masucci and Schiefelbeim, 1996; Zhang et al., 2005) and cucumber (Pierik et al., 1999). Recently, an involvement of ethylene in the expression of CsIRT1, CsFRO2, and CsHA1 has been suggested. The expression of these genes was found to be increased by ethylene in Fe-deficient plants and to be reduced upon exposure to ethylene inhibitors (Waters et al., 2007). However, ethylene alone could not induce the Fe deficiency responses in the presence of iron. Nitric oxide (NO) has recently been proposed as a modulator of the iron stress responses in tomato (Graziano and Lamattina, 2007). Nitric oxide is produced in the root epidermis upon Fe deficiency and scavenging of NO was shown to prevent the expression of both the formation of root hairs and the expression of genes involved in iron acquisition. Similar to what have been reported for ethylene, application of exogenous NO was only effective in iron-deficient plants, suggesting the existence of either a repressive signal in the presence of iron or the necessity of a promotive signal, which is only produced in the absence of iron. On the contrary, bicarbonate has been shown to induce Fe chlorosis by inhibiting the expression of CsIRT1, CsFRO2, and CsHA1 in Fe-deficient plants, probably through alteration of a FIT-like transcription factor (Lucena et al., 2007). No regulatory mechanism specific to the acidification response has been described until now.
In conclusion, it is demonstrated here that the iron acquisition response in cucumber roots consists of dramatic changes in gene expression and in the structure of root epidermal cells. Based on the marked increase in transcript abundance of genes involved in iron acquisition in root epidermal cells of the sub-apical region of the roots, it can be assumed that the interaction of root cells with the rhizosphere occurs predominantly at the outermost cell layer of the root. In addition, single cell type-specific gene expression analyses proves to be an useful tool to study differences in the functions of related genes with similar expression pattern. This is shown in the case of CsHA2, which, unlike CsHA1, does not appear to contribute to the stress-induced rhizosphere acidification. Moreover, it is shown here that the LMPC-assisted isolation of cells can be applied to plants exposed to various stresses, setting the stage for global expression analysis in single cells or tissue types of the root subjected to several nutritional stresses.
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Acknowledgements |
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We would like to thank Professor Stefano Gustincich and his group (Laboratory of Neurogenomics, SISSA, Trieste, Italy) and Dr Chung-Wu Lin (NTU Center for Genomic Medicine, NTU, Taipei, Taiwan) for the use of the LMPC system and technical advice. We also thank Drs Paola Beraldo and Eugenio Pittioni (Department of Animal Science, University of Udine, Udine, Italy) and Drs Mei-Chu Chung and Paula Jay Perry (IPMB, Academia Sinica, Taipei, Taiwan) for technical advice and useful suggestions. We would also like to thank Thomas Ju Way Yang for critical comments on the manuscript. SS was supported by Area Science Park (Trieste, Italy) with an FVG-D4 Project grant. This work was partly funded by an Academia Sinica grant to WS.
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