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First published online September 30, 2005; 10.1105/tpc.105.035394 © 2005 American Society of Plant Biologists
RNA Interference Identifies a Calcium-Dependent Protein Kinase Involved in Medicago truncatula Root Development
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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). In roots, a Ca2+ gradient localized in root hair tips appears to be required for root hair elongation in Arabidopsis thaliana (Wymer et al., 1997). Ca2+ likely plays an important role in root cortical cell elongation as well (Cramer and Jones, 1996; Demidchik et al., 2002), but its involvement is less well documented than is the case for root hair development.
Roots are also the site of important symbiotic associations with mycorrhizal fungi that greatly facilitate phosphate uptake in most plants (Harrison, 1999) and with rhizobial bacteria that supply available nitrogen to some plants, most notably the legumes (Vance, 2001). In legumes, both a rapid Ca2+ influx and a subsequent Ca2+ spiking in root hairs exposed to rhizobia encode informational signals that are associated with the establishment of symbiosis (Ehrhardt et al., 1996; Oldroyd and Downie, 2004). The symbiotic Ca2+ response and associated root hair deformation occur only in root hairs that are at a specific developmental stage (Gage, 2004), suggesting an important link between root and symbiotic development. Furthermore, the product of the DMI3 (for Doesn't Make Infections3) gene, which encodes a Ca2+/calmodulin-dependent protein kinase (CCaMK), is required for both rhizobial and mycorrhizal symbioses (Levy et al., 2004; Mitra et al., 2004). However, dmi3 mutants have no reported visible root phenotype, indicating that the encoded DMI3/CCaMK protein is not a key player in root development.
In spite of the accumulating data correlating Ca2+ with root cell development, the actual roles played by Ca2+ in this process remain largely unknown because few of the components of the Ca2+ sensing/signal-transducing system have been identified. Ca2+ has a dual role in roots, being an important nutrient that is actively acquired by the root system and translocated to the shoot via the xylem as well as participating in signaling (White and Broadley, 2003). Identifying and functionally characterizing the components of Ca2+ signaling networks that are involved in root development are necessary to fully understand this process.
Plants possess several classes of Ca2+ binding sensor proteins, including calmodulins, calcineurin B–like proteins, CCaMK, and Ca2+-dependent protein kinases (CDPKs). Although the functions of most Ca2+ sensor proteins are still obscure and none has been implicated in root development, several CDPKs are known to have roles in defense, development, and adaptation to cold, drought, and salt stress (Cheng et al., 2002; Harper et al., 2004; Ludwig et al., 2004).
RNA interference (RNAi) in Agrobacterium rhizogenes–transformed roots has been shown to be an effective tool to study the function of individual genes involved in root biology and symbiosis (Limpens et al., 2003, 2004). Using an RNAi-based screen, we have identified a Medicago truncatula gene (CDPK1) predicted to encode a Ca2+-dependent protein kinase that has a critical role in root development. Silencing CDPK1 is also associated with significant diminution of both rhizobial and mycorrhizal symbiotic colonization. Based on these results and data gained from gene expression analyses and measurements of the accumulation of reactive oxygen species (ROS), we propose that CDPK1 is a key component of one or more signaling pathways that direct or modify cell expansion, cell wall synthesis, and the defense response, each of which may alter the efficiency of symbiotic colonization.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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; Harper et al., 2004) with unknown function. Additionally, we sequenced the CDPK1 gene (accession number AY823957) and found that the exon–intron structure (12 exons, 11 introns) is identical to that of Arabidopsis group IV CDPK genes (see Supplemental Figure 2A online). A search of the literature failed to reveal a function for any of these three Arabidopsis genes or homologous genes from other species. CDPK1 has a typical CDPK structure that includes a poorly conserved N-terminal region, followed by a kinase domain, an autoinhibitory/junction region, and four Ca2+ binding EF-hand motifs. DNA gel blot analysis (see Supplemental Figure 2B online) and searches of M. truncatula EST databases and the partially completed genomic sequence provided no evidence of additional CDPK1-like genes in the M. truncatula genome. Silencing of three additional M. truncatula CDPK-related genes (represented by EST accession numbers BM779695, AW775214, and BG644439) did not result in CDPK1 RNAi-like phenotypes (data not shown). Microarray expression analysis of CDPKi roots did not reveal changes in the expression of any of the four arrayed root-expressed CDPK-like genes that share at least some level of nucleotide similarity with CDPK1 (AW687350 [TC102855], 74% similarity over 150 nucleotides; AW736699 [TC108057], 77% similarity over 233 nucleotides; AL370721 [TC109054], 73% similarity over 140 nucleotides; and AL388430 [TC100954], 78% similarity over 90 nucleotides). When the pRNAi1444-2 construct, which contains 309 bp of CDPK1 derived from the 5' untranslated region and the adjacent sequence that encodes the poorly conserved N-terminal region, was used to generate transgenic roots, phenotypes identical to those of CDPKi roots were observed (data not shown). The N-terminal region of CDPKs is highly variable, and little or no sequence similarity in this region is observed even among closely related CDPKs. The identical RNAi-induced phenotype observed with the pRNAi1444-1 and pRNAi1444-2 constructs together with the above-mentioned data indicate that we are specifically silencing CDPK1.
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10 to 70% (average, 37% ± 7%; n = 138 roots examined) of that of control root hairs, a statistically significant difference. We also measured the length of 18 cortical cells in three zones (4, 8, and 12 mm from the root tip) from three independent control and CDPKi roots. This microscopic analysis revealed that CDPKi roots have short cortical cells (41.2 ± 9.3 µm in CDPKi roots versus 70.0 ± 17.4 µm in control roots), whereas the width of root cells was not affected (Figure 1E). A PROCDPK1–ß-glucuronidase (GUS) reporter construct introduced into M. truncatula roots indicated that the CDPK1 promoter is most active in the root elongation zone and in emerging and elongating root hairs (Figures 2A to 2D). This expression pattern correlates well with regions where phenotypic variations are observed.
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50% of infection sites (Figures 4C and 4D).
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). As expected, suppression of the EIN2-like gene in M. truncatula roots (EIN2i roots) led to short root hairs (see Supplemental Figure 3A online), whose lengths were on average 23% (n = 17 roots) of control hair lengths. However, in contrast with CDPKi roots, EIN2i roots nodulated efficiently (see Supplemental Figure 3B online). EIN2i roots were also longer than CDPKi roots, consistent with the reduced sensitivity of the roots to ethylene (see Supplemental Figure 3C online). These data suggest that the short root hair phenotype by itself cannot be responsible for the diminished ability of the CDPKi roots to nodulate.
To understand the progression of symbiotic signaling in CDPKi roots, we examined the activity of the ENOD11 (for Early Nodulin11) promoter (Journet et al., 2001). Expression of ENOD11, an early nodulation gene, in wild-type roots is activated when the roots are exposed to rhizobia or Nod factor and often serves as a marker indicating activation of the symbiotic signaling pathway. In CDPKi roots inoculated with rhizobia, PROENOD11-GUS reporter gene activity was similar to that found in inoculated control plants (data not shown), indicating that CDPKi roots likely have normal symbiont-stimulated signaling that is required for the induction of early nodulation gene expression (Catoira et al., 2000; Journet et al., 2001; Oldroyd and Downie, 2004). Thus, our data suggest that CDPK1 is not part of the symbiotic signaling pathway as defined by previously characterized genes such as DMI1, DMI2, and DMI3, mutation of which led to defective rhizobia-induced ENOD11 gene activation (Catoira et al., 2000). These data indicate a normal initial interaction of the CDPKi roots with rhizobia.
In addition to rhizobial symbiosis, legumes form a symbiosis with mycorrhizal fungi. Although the rhizobial and mycorrhizal symbioses are distinct in nature, their formation depends on overlapping signaling pathways (Harrison, 1999; Kistner and Parniske, 2002). We found that CDPKi roots were impaired in their ability to form a symbiotic association with Glomus versiforme. Although G. versiforme was able to form appressoria and enter the cortex, we observed only 25% of the number of infection events on CDPKi roots relative to the number on control roots. At 15 d after inoculation, CDPKi roots showed an average colonization level of 8.8% of root length, whereas controls showed an average of 34.2%. Once inside the cortex, the internal development of the fungus was dramatically altered in the CDPKi roots, with both the size and the morphology of the infection units noticeably different from those formed on control roots (Figure 5). In the CDPKi roots, intercellular hyphae grew through the cortex toward the inner cortical cell layers, but horizontal spread through the cortex was reduced. A comparison of the length of individual infection units revealed that those on the CDPKi roots were much shorter (527 ± 214 µm; n = 26) than those on control roots (1571 ± 726 µm; n = 32). Furthermore, the morphology of the fungus was different in the CDPKi roots in that the development of arbuscules, the site of nutrient transfer between symbionts, occurred only rarely (Figure 5).
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-fucosidase, pectinesterase, ß-1,3-glucanase, and peroxidase, each of which is involved in cell wall modification and can affect cell wall extensibility (Christensen et al., 1998; Micheli, 2001; Scheible and Pauly, 2004), was altered in CDPKi roots compared with control roots.
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Among the most highly overexpressed defense-related genes were those putatively encoding allene oxide cyclase, polygalacturonase inhibitor, and several chitinases and lipoxygenases, leading to the possibility that the defense response in CDPKi roots is constitutively active (see Supplemental Tables 1 and 2 online). The expression of several genes encoding proteins involved in hormone signaling and phenylpropanoid and isoprenoid metabolism was also perturbed in CDPKi roots (Figure 6A; see Supplemental Tables 1 and 2 online).
CDPK1 Expression
RNA gel blot experiments indicate that CDPK1 is moderately expressed in flowers, stems, leaves, and roots (Figure 7A), suggesting that this gene functions in organs other than roots. Application of the plant growth regulators 2,4-D, 1-aminocyclopropane-1-carboxylic acid (an ethylene precursor), and abscisic acid in amounts that are physiologically effective did not affect CDPK1 expression (Figure 7B). Wounding, but not desiccation, significantly induced the accumulation of CDPK1 transcripts in roots (Figure 7B). This result is consistent with a possible role of CDPK1 in the defense-related response, as indicated by microarray analysis. A PROCDPK1-GUS construct introduced into roots indicated that the gene's promoter is responsive to mechanical wounding (Figure 7C). Inoculation of roots with S. meliloti did not result in any significant modulation of CDPK1 transcript levels or any detectable changes in GUS reporter gene activity (data not shown).
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). Therefore, we examined ROS levels in CDPKi roots. ROS accumulation was measured in root segments by fluorescence measurement as described previously (Shaw and Long, 2003). These measurements suggested that CDPKi roots contained increased levels of ROS relative to control roots (Figure 8A) and are consistent with the increased expression of defense-related genes.
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60% of CDPKi root hairs, including almost all of those with abnormal structure, did not show a typical ROS gradient, although they did maintain a relatively high concentration of ROS within the cell (Figures 8B and 8C, insets). These data show that suppression of CDPK1 alters ROS accumulation in roots and root hairs and suggest an interaction between CDPK1-mediated Ca2+ signaling and ROS accumulation during root development.
The Actin Cytoskeleton Is Altered in CDPKi Root Cells
Actin is an essential component of the cytoskeleton and controls plant cell extension and tip growth (Vantard and Blanchoin, 2002). The F-actin network in root hairs is required for root hair elongation, and application of actin-depolymerizing drugs leads to the inhibition of hair elongation and tip swelling (Ketelaar et al., 2003), a phenotype often observed in CDPKi roots. To analyze the organization of the actin cytoskeleton in CDPKi roots, we stained F-actin with fluorescently labeled phalloidin (Alexa Fluor 488). In contrast with the discrete actin filament network observed in control root hairs, we were unable to detect long discrete actin cables in very short and deformed CDPKi root hair cells (Figures 9A to 9D). The strongest fluorescence in these CDPKi root hairs localized in foci distributed along the hairs, which suggests the accumulation of numerous short microfilament fragments. In CDPKi root hairs with a less severe phenotype, we observed F-actin organization more similar to that in control hairs (data not shown). Similar observations were made when epidermal root cells were examined. Many CDPKi epidermal cells showed diffuse fluorescence with occasional foci of bright fluorescence, which was rarely observed in control roots (Figures 9E and 9F).
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DISCUSSION |
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; Muller and Schmidt, 2004). Recent studies have identified several genes that are involved in root cell elongation. The identity and function of these genes provide a molecular basis that supports the importance of ROS, phospholipids, and hormone signaling as well as a dynamic cytoskeletal network and cell wall biosynthesis in root cell elongation (reviewed in Ueda et al., 2005).
In this study, we have demonstrated that CDPK1 is required for the normal development of hairy roots in M. truncatula. A role for Ca2+ signaling in the polar growth of root hairs is widely accepted. However, much less is known about the role of Ca2+ in cortical cell elongation. Interestingly, CDPK1 appears to be an important signaling protein in both root hair and cortical root cell elongation. The fact that CDPKi roots have altered expression of cell wall genes and ROS accumulation leads us to consider that these processes are directly or indirectly regulated by a CDPK1 signaling pathway that, at least to some extent, mediates cell wall development. Increased deposition of lignin found in CDPKi roots supports the prediction that cell wall composition has been modified. These data parallel the work of Cano-Delgado et al. (2003), who found that increased lignification, resulting from decreased cellulose synthesis, correlated with reduced cell elongation and activation of a defense response in Arabidopsis. Alternatively, the alterations observed in CDPKi cell wall composition partially resemble those seen in secondary cell wall development, implying that these cells have rapidly matured.
Changes in the expression of defense-related genes in CDPKi roots as well as the activation of CDPK1 gene expression upon wounding indicate that this gene may play a role in plant defense. However, our results do not necessarily provide evidence for the direct involvement of CDPK1 in the defense response. Although CDPK1 gene expression is induced by wounding, suggesting that it may be involved in the induction of the defense response, suppression of CDPK1 expression does not lead to the suppression of defense response genes, many of which are induced in CDPKi roots. Among the differentially regulated defense response genes are many that are involved in cell wall structure. It is known that cell wall modifications can activate the defense response, which, in turn, can alter cell wall composition and structure. This complicated interplay between plant defense and cell wall structure confounds a simple interpretation of our results.
Based on our observations, we considered three possible explanations for the decreased symbiotic colonization observed in CDPKi roots: the effect of root hair length and growth, either of which may affect the efficiency of rhizobial infection; changes in the structure of cell walls that may affect the symbionts' ability to penetrate host cells and spread through the root cortex; and the inability of the roots to suppress the defense response during the early stages of symbiotic interactions. When we silenced an EIN2-like gene, we demonstrated that short root hairs, by themselves, do not prevent efficient nodulation by rhizobia. Furthermore, root hairs are not thought to be important in mycorrhizal colonization; therefore, the presence of short root hairs on CDPKi roots probably does not account for the altered interaction with G. versiforme.
The cell wall constitutes a major obstacle in the establishment of root endosymbiosis, and one proposed strategy to overcome this barrier involves its remodeling at the site of symbiont entry (Brewin, 2004). G. versiforme enters the root and spreads through the cortex by a mechanism that is different from rhizobial invasion; however, altered cell wall composition may affect the efficiency of both types of symbiotic colonizations. Experiments with G. versiforme demonstrated that fungal colonization was diminished at several stages of infection in CDPKi roots. Altered cell wall composition or cytoskeleton organization may lead to reduced spread of hyphae through the cortex, entry into plant cells, and formation of arbuscules. The efficient progression of infection threads induced by rhizobia through the root cortex also requires modification of the cell wall and is associated with changes in polarization and cytoskeletal rearrangements in the outer cortical cells (Timmers et al., 1999), events that also occur during nonsymbiotic root development, in which Ca2+ signaling may also play an important role.
An altered defense response is another factor that could affect the efficiency of both bacterial and fungal symbiosis. It is thought that to achieve efficient colonization of plant roots, symbionts may temporarily suppress the plant's defense response (Garcia-Garrido and Ocampo, 2002; Mithofer, 2002). Microorganism-induced modification of cell walls may initiate plant defense signaling (Schulze-Lefert, 2004). Such defense signaling is usually accompanied by rapid changes in the expression of defense-related genes and the accumulation of phenolics and ROS. Increased expression of a subset of defense-related genes and accumulation of ROS in CDPKi roots may indicate a constitutively active defense signaling that leads to reduced symbiotic efficiency. We speculate that CDPK1 may be involved in the transient regulation of localized ROS accumulation in root cells at a particular stage of development or in response to specific environmental signals. Consistent with this possibility, CDPKs have been shown to be involved in the defense response and are also involved in the modification of NADPH oxidase activity (Romeis et al., 2000; Xing et al., 2001; Cheng et al., 2002; Ludwig et al., 2004). Furthermore, ROS and Ca2+ signals can operate in tandem and have been implicated in root hair elongation, the defense response, and cell wall biosynthesis (Foreman et al., 2003; Rentel and Knight, 2004).
We cannot exclude the possibility that CDPKi roots mature faster than control roots and that this leads to the altered expression of those defense-related genes that are normally expressed in older plant tissues. Such changes in the developmental dynamics of CDPKi roots may interfere with symbiotic interaction, leading to reduced efficiency of symbiosis. This would be consistent with the lower symbiotic efficiencies found in older root sections.
Calcium influxes are often correlated with actin reorganization during polar growth (Vantard and Blanchoin, 2002). The link between cytoplasmic calcium concentration and actin dynamics is not well understood, but several actin binding proteins, such as profilin, villin, and actin-depolymerizing factor (ADF), are proposed to be downstream effectors of calcium-induced actin reorganization (Staiger, 2000). ADF induces the depolymerization of actin filaments by accelerating their rate of depolymerization (Pollard et al., 2000). Phosphorylation of ADF inhibits its interaction with both actin monomers and actin filaments (Smertenko et al., 1998). A calcium-dependent protein kinase-like activity has been implicated in the phosphorylation of plant ADF (Allwood et al., 2001), possibly connecting Ca2+ signaling to the dynamics of the actin cytoskeleton. The fact that CDPKi roots show altered actin cytoskeleton organization indicates that CDPK1 may be directly or indirectly involved in actin cytoskeleton modification.
Our results imply that inactivation of CDPK1 affects actin cytoskeleton organization, the accumulation of ROS, and the expression of genes involved in cell wall composition, defense, and hormone metabolism. Each of these processes may contribute to the observed CDPKi root developmental and symbiotic phenotypes. Further study is needed to clarify which processes are regulated directly by the CDPK1-mediated signaling pathway(s) and which are subject to indirect modification.
In the future, it would be interesting to explore the possible connection between the CDPK1-mediated signaling pathway and the pathways mediated by ROS-activated Oxi1 kinase and SIMK (for Stress-Inducible Protein Kinase), which are known to be important for both root hair development and defense (Samaj et al., 2002; Rentel et al., 2004), and to examine the possible interaction of CDPK1 with actin binding proteins. Further analysis of the molecular function of CDPK1 and dissection of the CDPK1-mediated signaling network will facilitate our understanding of signaling during root development.
In this study, the involvement of CDPK1 in root development and symbiosis was revealed solely through an RNAi-based functional screen. RNAi has been used previously for the identification or validation of biological functions of individual genes or small groups of genes (Limpens et al., 2003; Waterhouse and Helliwell, 2003). However, in this report, we identify gene function in plant roots as a result of a moderate-scale RNAi-based functional screen of genes with unknown function. Thus, an RNAi-based screen in plant roots may be compatible with large-scale functional gene analysis (Hilson et al., 2004). Another advantage of this approach as an alternative to screening mutants is that it can be used to study genes that have essential functions during embryonic development. We are currently trying to obtain stably transformed plants in which CDPK1 has been silenced. Such plants may further facilitate the analysis of CDPK1 function under different conditions, in various genetic backgrounds, and in nonroot tissues.
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METHODS |
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]) seeds were used for the generation of transgenic roots. Fragments of genes were amplified by PCR from cDNA clones and introduced into RNAi-inducing pHellsgate 8 vector (Helliwell et al., 2002) using the Gateway system (Invitrogen). To create the constructs that silence CDPK1, the regions corresponding to 265 to 678 and –24 to 285 nucleotides (relative to the ATG start codon) were amplified from AW775106 or AY821654 and introduced into pHellsgate 8, creating the CDPK1 pRNAi1444-1 and pRNAi1444-2 constructs, respectively. Roots generated with pRNAi1444-1 were named CDPKi roots and were used in all of the data presented. pEIN2i was created by introducing a fragment of the EIN2 gene (BG584510) into pHellsgate 8. The nonrecombinant pHellsgate 8 vector and pHellsgate 8 vector with a fragment of the human myosin gene were used as controls. The resulting recombinant constructs were introduced into Agrobacterium rhizogenes ARqua1 and used for plant transformation as described (Boisson-Dernier et al., 2001). Transgenic roots were selected on modified Farhaeus-agar medium supplemented with 22.5 to 27 mg/L kanamycin (Boisson-Dernier et al., 2001).
Rhizobial and Mycorrhizal Inoculation
For nodulation experiments, plants were inoculated with S. meliloti ABS7M on buffered nodulation medium agar plates (Ehrhardt et al., 1992) supplemented with 1 mM -aminoisobutyric acid or in Turface (Profile Products). Nodules were scored at 17 to 21 d after inoculation. Each transgenic plant had two to three independent transgenic roots. Remaining roots were removed.
For mycorrhizal experiments, transformed A17 seedlings were grown on a modified Fahraeus medium with 30 µM phosphate as described previously (Harrison et al., 2002; Liu et al., 2003). After 18 d on Fahraeus medium, plants were transferred to Turface and inoculated with 300 surface-sterilized G. versiforme spores as described previously (Harrison et al., 2002; Liu et al., 2003). Plants were harvested at 14 to 17 d after inoculation and stained with WGA-Alexa Fluor 488 (Molecular Probes) at a concentration of 0.2 µg/mL in PBS to visualize the fungus. Roots were viewed via fluorescence microscopy, and colonization was counted using the modified gridline intersect method (McGonigle et al., 1990). Measurements of the length of the infection units were made using Metamorph software (Universal Imaging). Three replicate experiments generated a total of 28 plants with CDPKi transgenic roots. Each plant contained two to three independent transgenic roots. The phenotype was consistent in all three experiments and visible at both times of harvest (14 and 17 d after inoculation).
Actin Cytochemistry
Root segments were fixed in 4% formaldehyde in ASB buffer (25 mM PIPES, pH 6.8, with 2 mM EGTA and 2 mM MgCl2) at room temperature for 1 h. The specimens were stained in Alexa Fluor 488 phalloidin (Molecular Probes; 0.66 µM in ASB buffer) at room temperature for 3 h and observed by fluorescence microscopy.
Cloning and Sequencing
Genomic DNA from A17 was digested with BamHI and cloned into 
DASH II (Stratagene). A positive clone with a 15-kb insert was identified by screening the library with labeled cDNA derived from AW775106 and used for further characterization. A 15-kb fragment from the DASH II clone was subcloned into pBluescript SK+ (Stratagene) and sequenced. Analysis indicated that the gene is composed of 12 exons that encode a 560–amino acid polypeptide. A cDNA containing the entire CDPK1 coding region was cloned and sequenced after cDNA synthesis and amplification using PfuUltra high-fidelity DNA polymerase (Stratagene). A 2300-bp fragment containing the promoter region and the first three nucleotides of the CDPK1 coding sequence was introduced into the pBI101 vector (Clontech), which contains a GUS gene.
RT-PCR and Expression Analysis
Total RNA was extracted from individual or bulked hairy roots (20 to 25 individual roots) from plants growing on agar plates at 22°C and a 16-/8-h light/dark photoperiod using TRIzol reagent (Invitrogen). Genomic DNA was removed using a DNA-free kit (Ambion). cDNA was synthesized using Superscript II reverse transcriptase (Invitrogen) and used for semiquantitative RT-PCR with the Taq PCR core kit (Qiagen) or for microarray experiments. The following primers were used for semiquantitative RT-PCR of CDPK1 transcripts: P1, 5'-CACCAATCATCTCACACGCTTTCC-3'; P2, 5'-GGGAAATTGTTGGGTCATGG-3'; P3, 5'-GGTCTAGTAGTTCTCCACCTTCG-3'; P4, 5'-GATGCCTCTCCTCCTTCTCTAAC-3'. Primers P2 (sense) and P3 (antisense) were designed within the region used for the RNAi construct, and primers P1 (sense) and P4 (antisense) were designed upstream and downstream, respectively, of the region used for the RNAi construct. Amplification of actin genes was used as a control with primers 5'-GGCATCACTCAGTACCTTTCAACAG-3' and 5'-CCAAAGCATCAAATAATAAGTCAACC-3'.
Microarray Data Analysis
We used the long oligonucleotide array (70-mer; Qiagen Medicago Genome Oligo Set version 1.0) designed based on tentative consensus sequences from The Institute for Genomic Research (Quackenbush et al., 2000; Quackenbush, 2001). RNA was isolated from CDPKi and control roots separately from three independent transformation experiments (biological replicates), each consisting of 20 to 24 transgenic roots. cDNAs were synthesized from 8.25 µg of total RNA for each biological replicate with a RT primer having a capture sequence for either the Cy3 or Cy5 dye molecule using a 3DNA Array 900 expression array detection kit according to the manufacturer's instructions (Genisphere). In total, six slides were used (two slides for each dye swap per biological replicate). The hybridization and washing procedures of the 3DNA Array 900 expression array detection kit were followed. Microarray slides were scanned using a two-laser scanner (GenePix 4000B scanner from Axon Instruments), and image analysis was performed using GenePix software (Axon Instruments). Background-subtracted mean intensities for both tissues were log transformed and normalized before further analysis. Within-slide normalization was performed using local linear regression (LOWESS function) (Yang et al., 2000), followed by between-slide normalization using four-way analysis of variance with replications for multislide dye-swap experiments (Kerr et al., 2000). Data were analyzed only for features with no missing data or with one missing data point for one dye-swap replicate. After data normalization, identification of differentially expressed genes was done using the statistical analysis of microarrays (Tusher et al., 2001). To detect differentially expressed genes and to eliminate those that have inconsistent expression data in replicated experiments, we used statistical analysis of microarrays, which allows the estimation of the false discovery rate for multiple testing (Tusher et al., 2001). A delta criterion that allowed a false discovery rate of <0.5% was applied. Genes that satisfied the statistical threshold and had a 1.8-fold change in expression were identified as upregulated or downregulated in CDPKi roots. Detailed data are presented in the supplemental data online.
ROS Measurement and Imaging
Measurement of ROS in roots was performed as described by Shaw and Long (2003) using the Amplex red hydrogen peroxide/peroxidase assay kit (Molecular Probes). Twelve to 15 roots were used for each experiment (two replicates). To image ROS accumulation in roots, roots of 12-d-old plants were incubated for 2 h at 4°C with 10 µM CM-H2DCF diacetate acetyl ester (Molecular Probes). Roots were excised, washed several times with water, and immediately used for imaging. Imaging analysis was performed with a Bio-Rad 1024 confocal system using 488-nm photons for dye excitation and detecting photons emitted at 522 nm. Typically, 30 to 40 sections of the Z-stack were collected using 10% of laser power and projected to generate a final image.
Accession Numbers
Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AY821654 and AY823957.
Supplemental Data
The following materials are available in the online version of this article.
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Acknowledgments |
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Footnotes |
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The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: J. Stephen Gantt (gantt001{at}tc.umn.edu).
Online version contains Web-only data.
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.105.035394.
Received June 21, 2005; Revision received August 6, 2005. accepted September 6, 2005.
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