TY - JOUR
AU - Gantt, J. Stephen
AB - Abstract Changes in cellular or subcellular Ca2+ concentrations play essential roles in plant development and in the responses of plants to their environment. However, the mechanisms through which Ca2+ acts, the downstream signaling components, as well as the relationships among the various Ca2+-dependent processes remain largely unknown. Using an RNA interference–based screen for gene function in Medicago truncatula, we identified a gene that is involved in root development. Silencing Ca2+-dependent protein kinase1 (CDPK1), which is predicted to encode a Ca2+-dependent protein kinase, resulted in significantly reduced root hair and root cell lengths. Inactivation of CDPK1 is also associated with significant diminution of both rhizobial and mycorrhizal symbiotic colonization. Additionally, microarray analysis revealed that silencing CDPK1 alters cell wall and defense-related gene expression. We propose that M. truncatula CDPK1 is a key component of one or more signaling pathways that directly or indirectly modulates cell expansion or cell wall synthesis, possibly altering defense gene expression and symbiotic interactions. INTRODUCTION Plant roots are required for mineral and water uptake as well as anchoring plants to soil. In response to numerous biotic and abiotic signals, root growth and development are modulated through cell expansion and division, cell growth polarity, and cell wall biosynthesis. These responses are subject to genetic and hormonal controls and are initiated by activating specific signaling pathways. Ca2+ is a ubiquitous secondary messenger, and changes in cytosolic Ca2+ concentration are associated with plant growth, development, and stress responses (White and Broadley, 2003). 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. RESULTS CDPK1 Functions in Root Development To identify components of root development and symbiotic signaling, we used RNAi to screen 154 root-expressed genes for their involvement in root development and symbiosis. Candidate genes for this screen were selected based on their expression profiles obtained from M. truncatula EST library mining and microarray data as well as their predicted functions. Most of the targeted genes (89%) are thought to be involved in signal transduction, and 81 of the 154 genes were putative protein kinases. Although silencing the expression of several of these genes gave easily visible phenotypes (S. Ivashuta and J.S. Gantt, unpublished data), one particular gene (CDPK1), predicted to encode a CDPK, was chosen for in-depth analysis. The RNAi-inducing construct pRNAi1444-1, which was designed to silence CDPK1 gene expression, contained sequences partially encoding the putative kinase domain. Composite plants whose roots have been transformed by A. rhizogenes carrying the pRNAi1444-1 plasmid (henceforth termed CDPKi roots) have stunted roots and short root hairs (Figures 1A and 1B Figure 1. Open in new tabDownload slide Characterization of M. truncatula CDPK1-Silenced (CDPKi) Roots. Comparisons of control (left) and CDPKi (right) roots are shown in (A) to (D). (A) and (B) Suppression of CDPK1 gene expression results in short roots (A) and short root hairs (B). Bar = 100 μm. (C) RT-PCR analysis shows significant suppression of CDPK1 gene expression in CDPKi roots. Primers P2 (sense) and P3 (antisense) are within the region of the gene used for the pRNAi1444-1 construct, and primers P1 (sense) and P4 (antisense) are upstream and downstream of this region, respectively. (D) Root hair phenotypes of control and CDPKi. Bars = 50 μm. (E) Cortical root cell elongation is affected in CDPKi. Bars = 30 μm. Figure 1. Open in new tabDownload slide Characterization of M. truncatula CDPK1-Silenced (CDPKi) Roots. Comparisons of control (left) and CDPKi (right) roots are shown in (A) to (D). (A) and (B) Suppression of CDPK1 gene expression results in short roots (A) and short root hairs (B). Bar = 100 μm. (C) RT-PCR analysis shows significant suppression of CDPK1 gene expression in CDPKi roots. Primers P2 (sense) and P3 (antisense) are within the region of the gene used for the pRNAi1444-1 construct, and primers P1 (sense) and P4 (antisense) are upstream and downstream of this region, respectively. (D) Root hair phenotypes of control and CDPKi. Bars = 50 μm. (E) Cortical root cell elongation is affected in CDPKi. Bars = 30 μm. ). The 560–amino acid sequence derived from the cloned full-length CDPK1 transcript (accession number AY821654) shares extensive sequence similarity (see Supplemental Figure 1 online) with CDPKs from several plant species, including CPK16, CPK18, and CPK28 of Arabidopsis, which are all members of a distinct CDPK group (group IV) (Harmon et al., 2001; 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. RT-PCR analysis of RNA isolated from CDPKi roots revealed a decrease in CDPK1 expression compared with that in control roots (Figure 1C). RT-PCR analysis was performed on RNA isolated from eight individual roots and on RNA isolated from two independent pools of control and CDPKi roots (24 roots in each pool) and always revealed a considerable (8- to 50-fold) reduction in CDPK1 transcripts in roots displaying the typical CDPKi phenotype (data not shown). However, because the severity of the phenotype varied and depended on the roots' developmental stage, it was difficult to phenotypically categorize them into groups and correlate the phenotype with the level of gene suppression. The average root length of CDPKi plants was only 42% (n = 127) of that of control roots. The length of fully extended root hairs on these roots (Figure 1D) varied from ∼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–β-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 Figure 2. Open in new tabDownload slide Roots Containing the PROCDPK1-GUS (CDPK-GUS) Reporter Gene Stained for GUS Expression. (A) to (C) A high level of promoter activity was detected in the root elongation zone (A) (bars = 150 μm) and in actively growing (B) and emerging (C) root hairs (bars = 10 μm) compared with the PROENOD11-GUS (ENOD11-GUS) reporter gene used as a control. (D) Portion of a root expressing the PROCDPK1-GUS reporter gene just above the root elongation zone. Bar = 80 μm. Figure 2. Open in new tabDownload slide Roots Containing the PROCDPK1-GUS (CDPK-GUS) Reporter Gene Stained for GUS Expression. (A) to (C) A high level of promoter activity was detected in the root elongation zone (A) (bars = 150 μm) and in actively growing (B) and emerging (C) root hairs (bars = 10 μm) compared with the PROENOD11-GUS (ENOD11-GUS) reporter gene used as a control. (D) Portion of a root expressing the PROCDPK1-GUS reporter gene just above the root elongation zone. Bar = 80 μm. ). This expression pattern correlates well with regions where phenotypic variations are observed. CDPK1 Expression Is Required for Efficient Microbial and Fungal Symbiosis In addition to decreased root hair length, up to 60% of CDPKi root hairs developed abnormally. These abnormalities (Figure 3A Figure 3. Open in new tabDownload slide Abnormalities in Root Hair Development Caused by Decreased Expression of CDPK1. (A) Fully elongated root hairs on CDPKi roots. Enlargements of representative root hairs are shown in separate panels at right. Bar = 30 μm. (B) Untreated (−Nod) and Nod factor–treated (+Nod) actively growing root hairs on control roots (see Methods). For Nod factor treatment, roots were incubated for 36 h with 10 nM Nod factor diluted in water. Bars = 30 μm. Figure 3. Open in new tabDownload slide Abnormalities in Root Hair Development Caused by Decreased Expression of CDPK1. (A) Fully elongated root hairs on CDPKi roots. Enlargements of representative root hairs are shown in separate panels at right. Bar = 30 μm. (B) Untreated (−Nod) and Nod factor–treated (+Nod) actively growing root hairs on control roots (see Methods). For Nod factor treatment, roots were incubated for 36 h with 10 nM Nod factor diluted in water. Bars = 30 μm. ), such as root hair tip swelling, growth redirection, and branching, resemble deformations that normally occur in wild-type competent root hairs exposed to rhizobia or Nod factor (Figure 3B). Only 2 of 23 CDPKi roots inoculated with Sinorhizobium meliloti developed nodules when grown for 3 weeks on the surface of buffered nodulation medium agar medium. By contrast, all control roots developed nodules (14 ± 8 per plant; n = 16) under the same conditions (Figures 4A and 4B Figure 4. Open in new tabDownload slide Suppression of CDPK1 Gene Expression Resulted in Reduced Efficiency of Nodulation. (A) and (B) Control (A) and representative CDPKi (B) roots inoculated with S. meliloti and grown for 3 weeks on buffered nodulation medium supplemented with 1 mM α-aminoisobutyric acid, an ethylene inhibitor that promotes nodulation. Bars = 300 μm. (C) and (D) Control (C) and CDPKi (D) roots inoculated with S. meliloti carrying a LacZ reporter gene and grown on Turface and stained with X-Gal at 7 d after inoculation. Figure 4. Open in new tabDownload slide Suppression of CDPK1 Gene Expression Resulted in Reduced Efficiency of Nodulation. (A) and (B) Control (A) and representative CDPKi (B) roots inoculated with S. meliloti and grown for 3 weeks on buffered nodulation medium supplemented with 1 mM α-aminoisobutyric acid, an ethylene inhibitor that promotes nodulation. Bars = 300 μm. (C) and (D) Control (C) and CDPKi (D) roots inoculated with S. meliloti carrying a LacZ reporter gene and grown on Turface and stained with X-Gal at 7 d after inoculation. ). When grown in a solid medium (Turface), CDPKi roots developed fewer nodules than did control roots (39 ± 19 for control [n = 24]; 9 ± 6 for CDPKi [n = 15]). Furthermore, most nodules that did develop on CDPKi roots were found on roots with a less severe root hair phenotype. We did not observe a dramatic difference in the number of infection events in CDPKi roots relative to control roots. Although S. meliloti (ABS7M) is able to infect CDPKi roots, the progression of infection threads through cortical cells is reduced in ∼50% of infection sites (Figures 4C and 4D). To investigate the possibility that the short root hair phenotype alone is capable of significantly diminishing the efficiency of nodulation, we created a RNAi construct that suppressed a M. truncatula homolog of the Arabidopsis EIN2 (for ETHYLENE INSENSITIVE2) gene (Guzman and Ecker, 1990). 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 Figure 5. Open in new tabDownload slide Mycorrhizal Colonization Is Diminished in CDPKi Roots. Control (left column) and CDPKi (right column) roots were inoculated with G. versiforme, harvested at 17 d after inoculation, and stained with WGA-Alexa Fluor 488 (Molecular Probes) to enable visualization of the fungus. Horizontal spread of intercellular hyphae through the cortex is reduced in CDPKi roots (top row) compared with control roots. An overlay of the bright-field and fluorescence micrographs is shown in the middle row. The bottom row shows close-up views of the infection sites. The arrowheads point to arbuscules, and the arrows point to intercellular hyphae. Bars = 50 μm. Figure 5. Open in new tabDownload slide Mycorrhizal Colonization Is Diminished in CDPKi Roots. Control (left column) and CDPKi (right column) roots were inoculated with G. versiforme, harvested at 17 d after inoculation, and stained with WGA-Alexa Fluor 488 (Molecular Probes) to enable visualization of the fungus. Horizontal spread of intercellular hyphae through the cortex is reduced in CDPKi roots (top row) compared with control roots. An overlay of the bright-field and fluorescence micrographs is shown in the middle row. The bottom row shows close-up views of the infection sites. The arrowheads point to arbuscules, and the arrows point to intercellular hyphae. Bars = 50 μm. ). 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). Silencing CDPK1 Alters the Expression of Genes Involved in Cell Wall Formation and Defense To better understand the CDPK1-mediated regulation of root development and symbiotic interactions, we compared global gene expression in CDPKi and control roots using a 16,000-element oligonucleotide microarray (see Supplemental Table 1 online). Genes that satisfied the statistical threshold and had 1.8-fold change in expression were identified as upregulated or downregulated in CDPKi roots. Seventy-five genes were overexpressed (see Supplemental Table 2 online) and 62 genes were underexpressed (see Supplemental Table 3 online) in CDPKi roots compared with control roots. Nearly 50% of the overexpressed genes and 9% of the underexpressed genes in CDPKi roots were related to cell wall biosynthesis and/or defense (Figure 6A Figure 6. Open in new tabDownload slide Cell Wall Composition and Gene Expression Are Altered in CDPKi Roots. (A) Classification of genes whose expression was altered in CDPKi roots. Ratios of CDPKi to control root gene expression were obtained from microarray experiments. (B) CDPKi roots contain increased levels of lignin (pink coloration). Control and CDPKi plants were stained with phloroglucinol, and representative roots are shown. Bar = 100 μm. (C) Micrographs of root sections taken under UV light to visualize cell wall autofluorescence. Arrows point to vascular bundles. Bars = 25 μm. Figure 6. Open in new tabDownload slide Cell Wall Composition and Gene Expression Are Altered in CDPKi Roots. (A) Classification of genes whose expression was altered in CDPKi roots. Ratios of CDPKi to control root gene expression were obtained from microarray experiments. (B) CDPKi roots contain increased levels of lignin (pink coloration). Control and CDPKi plants were stained with phloroglucinol, and representative roots are shown. Bar = 100 μm. (C) Micrographs of root sections taken under UV light to visualize cell wall autofluorescence. Arrows point to vascular bundles. Bars = 25 μm. ), indicating that these processes are affected in CDPKi roots. The abundance of transcripts putatively encoding α-fucosidase, pectinesterase, β-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. Cell wall composition and integrity are closely related to cell expansion and the defense response. Interestingly, we detected increased cell wall lignification in six of the eight CDPKi roots that were examined (Figure 6B). Additionally, all four sectioned CDPKi roots that were examined exhibited increased cell wall autofluorescence (Figure 6C), indicating that cell wall composition and/or structure are altered in CDPKi roots. 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 Figure 7. Open in new tabDownload slide Expression of CDPK1 in Different Plant Organs and in Response to Various Treatments. (A) CDPK1 expressed at similar levels in various plant organs. Seven micrograms of total RNA from flowers (F), stems (S), leaves (L), and roots (R) was used for RNA gel blot analysis. (B) Histogram showing levels of CDPK1 expression in response to treatment of roots with 2,4-D (50 nM), 1-aminocyclopropane-1-carboxylic acid (ACC; 5 μM), and abscisic acid (ABA; 10 μM) for 24 h; wounding (30 min); desiccation (∼30% of weight loss); and inoculation with S. meliloti (1, 3, and 48 h). Results of the RNA gel blot analyses were normalized to nontreated controls used in each experiment, and the ratio of expression in treated versus control roots is presented. NIH Image software (Scion) was used to analyze band intensity. (C) Activation of PROCDPK1-GUS reporter gene expression in roots by wounding. Figure 7. Open in new tabDownload slide Expression of CDPK1 in Different Plant Organs and in Response to Various Treatments. (A) CDPK1 expressed at similar levels in various plant organs. Seven micrograms of total RNA from flowers (F), stems (S), leaves (L), and roots (R) was used for RNA gel blot analysis. (B) Histogram showing levels of CDPK1 expression in response to treatment of roots with 2,4-D (50 nM), 1-aminocyclopropane-1-carboxylic acid (ACC; 5 μM), and abscisic acid (ABA; 10 μM) for 24 h; wounding (30 min); desiccation (∼30% of weight loss); and inoculation with S. meliloti (1, 3, and 48 h). Results of the RNA gel blot analyses were normalized to nontreated controls used in each experiment, and the ratio of expression in treated versus control roots is presented. NIH Image software (Scion) was used to analyze band intensity. (C) Activation of PROCDPK1-GUS reporter gene expression in roots by wounding. ), 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). CDPKi Roots Have Altered ROS Accumulation ROS and Ca2+ signals are both implicated in root hair development and in the defense response (Mori and Schroeder, 2004). 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 Figure 8. Open in new tabDownload slide ROS Accumulation Is Altered in CDPKi Roots. (A) ROS measurement (arbitrary units; se is shown as vertical bars) using the Amplex red hydrogen peroxide/peroxidase assay kit in 1-cm-long roots. (B) and (C) Confocal microscopy images of roots stained for ROS accumulation (CM-H2DCF imaging). The root hair initiation zone and root elongation zone of control (B) and CDPKi (C) are shown. Insets show representative root hairs. Bars = 20 μm. Figure 8. Open in new tabDownload slide ROS Accumulation Is Altered in CDPKi Roots. (A) ROS measurement (arbitrary units; se is shown as vertical bars) using the Amplex red hydrogen peroxide/peroxidase assay kit in 1-cm-long roots. (B) and (C) Confocal microscopy images of roots stained for ROS accumulation (CM-H2DCF imaging). The root hair initiation zone and root elongation zone of control (B) and CDPKi (C) are shown. Insets show representative root hairs. Bars = 20 μm. ) and are consistent with the increased expression of defense-related genes. Observation of ROS accumulation using the fluorescent dye CM-H2DCF also demonstrated increased levels of ROS in 8 of 11 tested CDPKi roots compared with control roots. Similar to PROCDPK1-GUS reporter gene activity (Figure 2A), this increase in ROS was especially prevalent in the root elongation and root hair initiation zones (Figures 8B and 8C). Unlike actively elongating control root hairs, most of which exhibited a tip-high ROS gradient, ∼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 Figure 9. Open in new tabDownload slide The Actin Cytoskeleton of Root Cells Visualized with Alexa Fluor 488 Phalloidin. (A) and (B) Control root hair tip (A) and mid region (B). Arrows point to large bundles of actin filaments. (C) and (D) Representative CDPKi root hairs with an extreme length phenotype. The bright fluorescence foci suggest the accumulation of short microfilament fragments (arrowheads). (E) and (F) Control (E) and CDPKi (F) root epidermal cells. Bars = 10 μm. Figure 9. Open in new tabDownload slide The Actin Cytoskeleton of Root Cells Visualized with Alexa Fluor 488 Phalloidin. (A) and (B) Control root hair tip (A) and mid region (B). Arrows point to large bundles of actin filaments. (C) and (D) Representative CDPKi root hairs with an extreme length phenotype. The bright fluorescence foci suggest the accumulation of short microfilament fragments (arrowheads). (E) and (F) Control (E) and CDPKi (F) root epidermal cells. Bars = 10 μm. ). 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). DISCUSSION Root cell elongation is one of the last steps in root cell development, which also includes fate-dependent cell differentiation, oriented cell division, and cell specification. Alteration in the extent of root cell elongation is an important mechanism by which plants adapt to different soil conditions, facilitate water and nutrient uptake, and respond to their biotic environment (Gage, 2004; 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. METHODS Plasmid Construction and Plant Transformation Medicago truncatula A17 or L416 (A17 containing a PROENOD11-GUS construct [Journet et al., 2001]) 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 α-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 λDASH 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 λDASH 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. Supplemental Figure 1. Amino Acid Sequence Comparisons and Functional Regions of CDPK Proteins. Supplemental Figure 2. CDPK1 Gene Structure. Supplemental Figure 3. Characteristics of EIN2i Roots. Supplemental Table 1. Transcript Profile Data Comparing CDPKi and Control Roots. Supplemental Table 2. Genes Significantly Overexpressed in CDPKi Roots. Supplemental Table 3. Genes Significantly Underexpressed in CDPKi Roots. ACKNOWLEDGMENTS This work was supported in part by Minnesota Experiment Station Grant MIN-71-045 (to J.S.G. and C.P.V.) and by National Science Foundation Grants DBI-0110206 (to D.R. Cook, K.A.V., M.J.H., C.P.V., and J.S.G.) and DBI-0421676 (to J.S.G., K.A.V., M.J.H., and C.P.V.). We thank N. Sharopova for help with the analysis of microarray data; J. Denarie, D. Barker, and P.M. Waterhouse for providing Nod factor, PROENOD11-GUS plants, and pHellsgate 8 vector, respectively; and the editor and reviewers for constructive suggestions. REFERENCES 1. Allwood E.G. Smertenko A.P. and Hussey P.J. ( 2001 ). Phosphorylation of plant actin depolymerising factor by calmodulin-like domain protein kinase. FEBS Lett. 499 , 97 – 100 . Crossref Search ADS PubMed 2. Boisson-Dernier A. Chabaud M. Garcia F. Becard G. Rosenberg C. and Barker D.G. ( 2001 ). 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Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.105.035394. © 2005 American Society of Plant Biologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model ( https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - RNA Interference Identifies a Calcium-Dependent Protein Kinase Involved in Medicago truncatula Root Development
JF - The Plant Cell
DO - 10.1105/tpc.105.035394
DA - 2005-11-02
UR - https://www.deepdyve.com/lp/oxford-university-press/rna-interference-identifies-a-calcium-dependent-protein-kinase-YWmOq0cTaP
SP - 2911
EP - 2921
VL - 17
IS - 11
DP - DeepDyve
ER -