JXB Advance Access originally published online on July 26, 2006
Journal of Experimental Botany 2006 57(12):2923-2936; doi:10.1093/jxb/erl052
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RESEARCH PAPER |
Loss of function of COBRA, a determinant of oriented cell expansion, invokes cellular defence responses in Arabidopsis thaliana
1Department of Forestry, Michigan State University, East Lansing, MI 48824, USA
2DOE-Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA
*To whom correspondence should be addressed. E-mail: hanky{at}msu.edu
Received 5 February 2006; Accepted 1 May 2006
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Abstract |
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An Arabidopsis T-DNA insertion mutant that results in complete loss-of-function of the COBRA gene has been identified. The COBRA gene encodes a putative glycosylphosphatidylinositol (GPI)-anchored protein that modulates cellulose deposition and oriented cell expansion in roots. The loss-of-function mutant allele (named ‘cob-5’) exhibits abnormal cell growth throughout the entire plant body and accumulates massive amounts of stress response chemicals such as anthocyanins and callose. To gain further insight into the mechanism by which COBRA affects cell growth and physiology, the whole-genome gene expression profile of cob-5 plants was compared with that of wild-type plants. Consistent with the mutant phenotype, many genes involved in anthocyanin biosynthesis were up-regulated in the cob-5 plants, whereas genes involved in cell elongation were down-regulated. The most striking feature of the gene expression profile of cob-5 was the massive and co-ordinate induction of defence- and stress-related genes, many of which are regulated by the plant stress signal jasmonic acid (JA). Indeed, the cob-5 plants over-accumulated JA by nearly 8-fold compared with wild-type plants. Furthermore, induction of cell elongation defects in conditional allele cob-3 plants triggers the expression of a defence-responsive gene. These results provide potential clues to the mechanisms by which plant cells initially perceive biotic stress at the cell surface.
Key words: cell elongation, cell wall, COBRA, defence, jasmonic acid, whole transcriptome
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Introduction |
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The primary cell wall of plants, which provides both physical strength and extensibility to permit cell growth, comprises cellulose microfibrils impregnated in the matrix of pectins, xyloglucans, hemicelluloses, and glycoproteins (for a review see Dhugga, 2001). Developmental regulation of cell expansion is directed by proteins that interact with and modulate the polysaccharide network of the cell wall (Roudier et al., 2002). Over 400 proteins are predicted to be located in the cell wall (Arabidopsis Genome Initiative, 2000). It has been reported that glycosylphosphatidylinositol (GPI)-anchored proteins are targeted to the plant cell surface and are are involved in extracellular matrix-remodelling and signalling in plants (Sherrier et al., 1999; Borner et al., 2002; Shi et al., 2003). COBRA is an extracellular GPI-anchored protein, point mutations (cob-1, cob-2, and cob-3) of which resulted in the failure of oriented cell expansion and cellulose deposition in root cells (Schindelman et al., 2001; Roudier et al., 2002). Roudier et al. (2005) described various phenotypes of a null mutant (cob-4) of COBRA that established a role in anisotropic expansion of most developing organs through its involvement in cellulose microfibril orientation.
Increasing evidence indicates that the cell wall plays an important role in host surveillance of pathogens and, furthermore, may be a source of signalling molecules that orchestrate plant defence responses to pathogen infection (Vorwerk et al., 2004). The structure of the cell wall appears to be dynamic, changing in response to developmental and environmental stimuli. For example, pathogen-induced reactive oxygen species catalyse reactions that increase the physical strength of the wall and, thereby, attenuate pathogen ingress (Lamb and Dixon, 1997). Recent genetic studies have provided concrete evidence for a link between cell wall structure and host defence. Both eli1 (ectopic lignin1; Cano-Delgado et al., 2000) and cev1 (constitutive expression of VSP1; Ellis and Turner, 2001) mutants were identified as different mutant alleles of CESA3, a cellulose synthase gene in Arabidopsis thaliana. These mutants showed a reduction in cellulose synthesis, resulting in abnormal cell expansion. The cev1 plants also showed enhanced resistance to pathogens (Ellis et al., 2002; Cano-Delgado et al., 2003), which was attributed to constitutive production of jasmonic acid (JA) and activation of JA-dependent defences. These results suggest that changes in cell wall integrity can act as a signal for the activation of defence responses (Vorwerk et al., 2004).
JA and its methyl ester, methyl jasmonate (collectively referred to as JAs), are fatty acid-derived compounds synthesized from linolenic acid via the octadecanoid pathway (Schaller, 2001). JA is well known to act as an important cellular regulator in diverse developmental processes, such as seed germination, root growth, fertility, and senescence, as well as a signal in plant defence and environmental stress responses (for reviews see Wasternack and Hause, 2002; Cheong and Choi, 2003; Devoto and Turner, 2003). It is generally believed that a complex network of signalling pathways involving JA, salicylic acid (SA), and ethylene (ET) regulate plant defence responses against various pathogens. In Arabidopsis, the JA response pathway plays a key role in defence against necrotrophic pathogens and herbivores, whereas the SA response pathway is involved in resistance to biotrophic and some necrotrophic pathogens. These two pathways influence and interact with each other in a complex manner that remains to be elucidated (Kunkel and Brooks, 2002; Devoto and Turner, 2003). Several studies support positive interactions between JA and ET signalling pathways. For example, expression of several JA-dependent defence genes (i.e. PDF1.2, THI2.1, HEL, and CHIB) requires the presence of a functional ET signalling pathway (Norman-Setterblad et al., 2000). Additional evidence for the co-ordinate regulation of JA and ET pathways comes from Arabidopsis microarray experiments (Schenk et al., 2000). The microarray analysis showed that nearly half of the genes whose expression was induced by ET were also induced by JA treatment, although it also revealed that JA and ET independently regulate the expression of separate sets of genes.
In this study, an Arabidopsis T-DNA insertion mutant of the COBRA gene was isolated and characterized. This loss-of-function mutant, designated cob-5, exhibited extreme defects in the directional cell elongation throughout the plant body. By using genomics and biochemical analysis, it was demonstrated that the massive and co-ordinate induction of defence- and stress-related genes in cob-5 plants, many of which are regulated by the plant stress signal jasmonic acid (JA), are correlated with hyper-accumulation of JA. Further analyses suggest that the plant cells recognize the cell elongation defects caused by the null mutation of COBRA as a signal of pathogen attack for adjusting cell growth.
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Materials and methods |
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Plant material
Arabidopsis thaliana, ecotype Columbia (Col-0), was used in all the experiments. Seeds of wild-type and T-DNA insertional mutants, were surface-sterilized by washing first with 70% ethanol for 2 min, with sodium hypochlorite for 30 min, and finally with sterile distilled water. Sterilized seeds were imbibed at 4 °C for 3 d in the dark and then grown on MS nutrient agar media containing 2% sucrose in a growth chamber (16/8 h light/dark) at 23 °C.
Histological analysis
For confocal laser scanning microscopy, the 10-d-old seedlings of wild-type Arabidopsis and cob-5 mutants were stained with 0.1% aqueous solution of Safranin O for 15 min. A Zeiss PASCAL confocal laser scanning microscope (Jena, Germany), with a 488 nm excitation mirror, a 560 nm emission filter, and a 505–530 nm emission filter, was used to record images. Image analysis was performed using a Laser scanning microscope PASCAL LSM version 3.0 SP3 software.
For scanning electron microscopy, the 10-d-old seedlings of wild-type Arabidopsis and cob-5 mutants were fixed at room temperature in 4% glutaraldehyde buffered with 0.1 M sodium phosphate (pH 7.4) for 2 h. Following a brief rinse in the buffer, samples were dehydrated in an ethanol series (25, 50, 75, and 95%) for 15 min at each gradation and in three changes of 100% ethanol. The dehydrated samples were dried in a critical-point dryer with liquid CO2 as a transitional fluid, after which the samples were coated with gold and examined in a scanning electron microscope (JSM-35C, JEOL, Japan).
For callose detection, the leaves of 10-d-old wild-type Arabidopsis and cob-5 mutants were cleared and rinsed in water and then stained for 30 min in 150 mM K2HPO4 (pH 9.5) containing 0.01% aniline blue (Adam and Somerville, 1996). Leaves were mounted in 50% glycerol and examined with a Leica DM RA2 microscope with an A4 fluorescence cube (Hauck et al., 2003).
Gene expression analysis using Affymetrix GeneChipR
For total RNA isolation, the 10-d-old seedlings of wild-type Arabidopsis and cob-5 mutants were grown for 10 d on agar plates (MS+2% sucrose+0.3% phytagel) under long-day conditions (16/8 h light/dark). The plant samples were pooled from several batches of plants to minimize a variation in gene expression patterns caused by a subtle change in environmental condition and harvested around 16.00 h. For reproducibility, all experiments were duplicated. All methods for the preparation of cRNA from mRNA, as well as the subsequent steps leading to hybridization and scanning of the Arabidopsis ATH1 Genome Array (Affymetrix, Santa Clara, CA), were performed as described previously (Ko et al., 2004; Ko and Han, 2004). The average difference and expression call, for each of the duplicated samples, was computed using Affymetrix GeneChip Analysis Suite version 5.0 with default parameters. The resulting hybridization intensity values (signal intensity) reflect the abundance of a given mRNA species relative to the total mRNA population and were used in all subsequent analyses. About 74.7% of the probe sets (16 881 genes) on the GeneChip were expressed or detectable (‘presence’ calling) in the 10-d-old wild-type seedlings. Functional classifications and annotations of selected genes were performed based on the information of TAIR (http://www.arabidopsis.org/) and Munich Information Center for Protein Sequences (http://mips.gsf.de/proj/thal/db/tables/tables_func_frame.html).
RNA extraction and northern blot analysis
Total RNA was extracted using the Trizol reagent method (Gibco-BRL, Gaithersburg, MD). For northern blot analysis, 10 µg of total RNA of each sample was denatured and separated using a 1% agarose–-formaldehyde gel. RNA was transferred onto a Hybond-N+ membrane (Stratagene, La Jolla, CA) by capillary action. Gene-specific probes were prepared by PCR and labelled with [
32P]-dCTP using a Prime-it II Random Primer Labeling kit (Stratagene, La Jolla, CA). Hybridization was carried out according to the manufacturer's instructions, and a Kodak Biomax film (Sigma) was exposed to the blot. Ethidium bromide-stained ribosomal RNA was used as a loading control.
PCR screening
To identify the T-DNA insertion site of SALK T-DNA insertional mutants, an adaptor-ligated PCR method was used (Alonso et al., 2003). Gene-specific forward primer (F; 5'-TCTTTCTTCTCCAGATCCACCT), reverse primer (R; 5'-GGCAGAGAAGAAGAAAAAGACA), and the left-border primer of T-DNA (TL; 5'-GCGTGGACCGCTTGCTGCAACT) were used for PCR-based genotyping. DNA fragments amplified with the gene-specific and left-border primers were sequenced to confirm the insertion sites.
Segregation analysis
Since celd1-1 homozygotes are lethal after seedling stage, celd1-1 heterozygotes were identified using PCR screening and self-pollinated. The ratio of wild type: celd1 plants was determined from F2 populations using four independent experiments.
Biochemical analysis
The anthocyanin and UV-absorptive compounds were extracted by incubating 10 seedlings of 10-d-old wild-type Arabidopsis and the cob-5 mutant in 300 µl of extraction solution (methanol+1% HCl) overnight at 4 °C. After the extraction, 200 µl of water and 200 µl of chloroform were added, and the mixture was centrifuged to remove the seedlings. The amount of anthocyanin was measured at A530 for cyanidines and A515 for pelagonidins. UV-absorptive compound was measured at A330. These analyses were performed using three independent samples.
JA measurements
Seedlings grown on agar plates (
100 mg of tissue) for 10 d were collected and immediately frozen in liquid nitrogen. Jasmonic acid (JA) was extracted and measured as described by Li et al. (2002). Dihydrojasmonic acid was added to samples as an internal standard. Methylated carboxylic acids from plant samples were volatilized and collected on volatile collection traps® (VCT) (Analytical Research Systems Gainesville, Florida). Samples were eluted from the VCT resin by methylene chloride and subsequently analysed by gas chromatography–mass spectroscopy (GC–MS). GC–MS analysis was performed by selected ion monitoring, with isobutane chemical ionization as described. The GC–MS system consisted of a 6890 Network GC connected to a 5973 inert Mass Selective Detector (Agilent, Palo Alto, CA, USA). Compounds were separated on a HP5MS column (30 m x 0.25 mm x 0.25 µm). The temperature regime for GC was 40 °C for 1 min after injection, followed by sequential temperature ramps of 25 °C min–1 to 150 °C, 5 °C min–1 to 200 °C, 10 °C ramp to 240 °C. The 240 °C temperature was maintained for 10 min. The identity of MeJA in plant samples was confirmed by comparison of the elution time and mass spectra to an authentic MeJA standard (Sigma).
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Results |
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A novel mutant exhibits dramatic alterations in plant growth and development
During a screen for T-DNA insertion mutants of Arabidopsis that are affected in the secondary growth (Ko et al., 2004), one mutant line was found, SALK_093560 (T-DNA Express, http://signal.salk.edu/cgi-bin/tdnaexpress), with an extreme dwarf phenotype, which was initially named celd1 (cell elongation defect1). Compared with the wild type, 10-d-old celd1-1 seedlings grown on MS agar medium containing 2% sucrose exhibited dwarfed aerial tissues as well as short and thick roots (Fig. 1A, B, C, D, E). Hypocotyls and petioles were shorter, and leaf blades were smaller than those of wild-type plants (Fig. 1F, G, H, I). In addition, most epidermal tissues of celd1-1 plants contained many swollen cells (Fig. 1C, G, I; Fig. 2D, F). Closer inspection of the epidermal cells of the petiole and hypocotyl tissues suggested that the celd1-1 mutation causes defects in longitudinal cell elongation and cell division (Fig. 2A, B, C, D). The surface of celd1-1 leaves lacked the typical epidermal pavement cells that were seen in wild-type plants (Fig. 2E, F). These observations indicate that the celd1 mutation disrupts a physiological process essential for normal plant growth and development.
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celd1 plants over-accumulate flavonoids and callose
Another overt phenotype of celd1 plants was the accumulation of purple-coloured pigments in the aerial body (Fig. 1G, I). Compared with the wild type, the amount of anthocyanins and UV-absorptive compounds in celd1-1 plants was increased by about 10-fold and 2-fold, respectively (Fig. 3A). celd1-1 plants were also assessed for the deposition of callose, another marker of plant stress (Jacobs et al., 2003). Aniline blue staining showed that deposition of callosic papillae (shown as bright speckles) were abundant in celd1-1 leaves, but were not detected in wild-type plants (Fig. 3B). These results indicate that celd1 mutants behave as if they were under a severe biotic and/or abiotic stress.
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celd1 is a null mutation of the COBRA gene
In an attempt to identify the gene responsible for the celd1-1 phenotype, it was not possible to confirm the original annotation that the SALK_093560 line contains a T-DNA insertion in At1g24500 (T-DNA Express; data not shown). To resolve this question, adaptor-ligated PCR (Alonso et al., 2003) was conducted to identify genomic fragments flanking the T-DNA insertion site. Two different sized PCR products were identified suggesting that the SALK_093560 line contains at least two T-DNA insertions. Sequence analysis of PCR products revealed that one T-DNA was inserted into the 8th exon of At1g77260 and the other into the 5th exon of COBRA (At5g60920). PCR-based genotyping of segregating populations allowed the lines that were homozygous for a T-DNA insertion into either At1g77260 or COBRA to be isolated; only the latter lines showed the celd1-1 phenotype (data not shown). To verify that disruption of COBRA is responsible for the celd1-1 phenotype, a second SALK line (SALK_051906) harbouring a T-DNA insertion at this locus, was identified and characterized. Homozygous lines for this insertion (designated as celd1-2) exhibited a morphological phenotype that was indistinguishable from celd1-1 (Fig. 4A, B).
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Anthocyanin accumulation of celd1-2 was also evident on seedlings 7 d after germination (data not shown). The insertion site and orientation of T-DNA in celd1-2 was confirmed by PCR (Fig. 4C). Northern blot analysis showed that expression of the CELD1/COBRA gene was abolished in the celd1-1 and celd1-2 mutants, indicating that they are null mutants (Fig. 4D). Segregation analysis of F2 populations derived from self-pollinated celd1-1 heterozygotes showed that the ratio of wild-type to celd1 plants was 389:125 (3.1:1).
2 value is 0.127 which is calculated based on a 3:1 ratio of wild-type to mutant plants. The critical value is 3.84 (P=0.05). This result indicates that the celd1 phenotype is inherited as a single recessive mutation (Fig. 4E). It was concluded that the celd1 phenotype results from loss-of-function of the COBRA gene. Hereafter, celd1-1 and celd1-2 were designated as cob-5 and cob-6, respectively, and the gene was referred to as COBRA.
Transcription phenotype the of cob-5 plant is highly correlated to its physiological phenotype
To gain further insight into the molecular mechanism of COBRA action, whole-genome transcript profiling was conducted of wild-type and cob-5 plants that were grown for 10 d on MS agar media containing 2% sucrose. Gene expression data were obtained from two independent experiments conducted with the Arabidopsis ATH1 Genome Array (Affymetrix, Santa Clara, CA), which represents 22 620 genes. The correlation coefficient values (R2) of each duplicated experiment in the wild type and cob-5 were 0.9646 and 0.9926, respectively, indicating the high reproducibility.
The expression profile of genes that are known to be involved in cell wall biogenesis was investigated. However, these genes showed no changes in expression level between wild-type and cob-5 mutants (see supplementary Table I at JXB online). This implies that the post-transcriptional regulation might be responsible for the low level of cellulose biosynthesis observed in cobra null mutants (Roudier et al., 2005). Since the most prominent phenotype of cob-5 was the abnormal cell shape (Fig. 2), the effect of the mutation on the expression of genes involved in cell elongation was determined. Cell elongation is dependent on cell wall relaxation and expansion driven by water influx into plant vacuolar compartments (McCann et al., 1993). The former is controlled by a series of cell wall-loosening/hydrolytic enzymes (Cosgrove, 2001) and the latter by the water channel protein aquaporin (Maurel and Chrispeels, 2001). Many expansin and aquaporin genes were significantly down-regulated in cob-5 plants (see supplementary Table II at JXB online). Six out of 23 expansin genes identified in this analysis were down-regulated more than 2-fold. Similarly, nine out of 24 aquaporin genes were down-regulated more than 2-fold. Genes encoding xylogucan endotransglycosylases, including XTR7 and XTR3, were also down-regulated up to 10-fold. Gibberellic acid (GA) is a well-known growth regulator that promotes cell elongation (for reviews see Kende and Zeevaart, 1997; Ross and O'Neill, 2001). Clear differences were observed in the expression of various GA biosynthetic genes. Specifically, GA20-oxidase genes (At3g46490 and At5g51810) were down-regulated by 6.7- and 2.2-fold, respectively, while a GA2-oxidase gene (At1g30040) was up-regulated by 6.8-fold, implying a possible reduction in active GAs in cob-5 plants (see supplementary Table II at JXB online). Most of the key regulatory genes of anthocyanin biosynthesis were highly up-regulated (see supplementary Table III at JXB online), which is consistent with the hyper-accumulation of anthocyanins in the cob-5 mutant plants (Fig. 3A). These results clearly show that the transcription phenotype of cob-5 plants is highly correlated with its physiological phenotype.
Loss of COBRA function results in up-regulation of defence-related genes
Functional classification of genes whose expression increased more than 3-fold in either the cob-5 or wild-type plants was performed (Fig. 5). A total of 499 genes were up-regulated in cob-5 plants, whereas 273 genes were down-regulated in comparison to the wild type (gene lists are on supplementary Tables IV and V at JXB online). A striking feature of the up-regulated genes was a high proportion (28.4%) of genes involved in defence and secondary metabolism (Fig. 5; Table 1). These genes can be classified into anti-microbial functions, transcription factors, disease resistance (R) genes, and components of the oxidative burst. Genes encoding transcription factors were selected based on their pathogen-inducibility data from previously reported microarray experiments (Tao et al., 2003). Five AP2/EREBPs and six WRKY transcription factors were up-regulated up to 13-fold. Genes involved in the biosynthesis of JA, SA, and ET were also listed because these signalling molecules have a pivotal role in defence signalling (for a review see Kunkel and Brooks, 2002). The cob-5 mutation appeared to have greatest impact on the expression of genes encoding JA biosynthetic enzymes. For example, genes encoding lipoxygenase (LOX), allene oxide synthase (AOS), and 12-oxophytodienoate reductase (OPR3) were up-regulated up to 8-fold. While modest changes were observed in the expression of genes involved in SA biosynthesis, genes participating in ET biosynthesis were unaffected by the mutation (Table 1). Consistent with the up-regulation of JA biosynthetic genes, the expression of JA-regulated defence genes such as plant defensin (PDF1.2), thionin (Thi2.1), VSP1, PR-3, and PR-4 was massively increased (up to 106-fold). In addition, many JA-responsive genes, such as those encoding trypsin inhibitors, myrosinase binding proteins, terpene synthase, tyrosine aminotransferase, AtEXT1, and AtCOR1, were also highly up-regulated in the cob-5 plants (Table 1).
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Of the 142 genes up-regulated (3-fold or higher) in cob-5 plants, 100 of these were previously shown to be up-regulated by the treatment with various strains of Pseudomonas syringae (Tao et al., 2003) (Table 2). This finding supports the general conclusion that loss of COBRA function results in constitutive expression of defence- and stress-related genes.
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In addition to the induction of JA-regulated genes in cob-5 plants, up-regulation of several SA-regulated genes was also observed in the mutant. These included PR-1, PR-2, PR-5, and AtWRKY70 (Li et al., 2004) as well as SA biosynthetic-genes such as PAD4, EDS1, and EDS5/SID1 (Table 1). In addition, 10 of the 100 genes up-regulated in both the cob-5 and the pathogen-treated plants (Table 2) seem to require SA because the expression of those genes was not changed in the NahG background that is deficient in SA accumulation (nahG) (Lawton et al., 1995).
cob-5 plants overproduce JA
The high level of expression of JA-responsive genes in cob-5 suggested that the mutant might constitutively accumulate JA. To test this hypothesis, the endogenous level of JA was measured by gas chromatography–mass spectroscopy (GC–MS). Results from three independent experiments showed that the level of JA in cob-5 seedlings grown on MS agar medium was 7.6-fold greater than that in wild-type control plants (Fig. 6).
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Induction of cell elongation defects in cob-3 triggers the expression of defence-responsive gene, PDF1.2a
The ‘conditional’ cob-3 missense mutant was used to test the link between cell elongation defects and activation of cellular defence responses further. Previous studies showed that the root phenotypes of cob-3 plants are dependent upon the amount of sucrose in the growth medium; plants grown on medium containing less than 2% sucrose appeared phenotypically normal (Benfey et al., 1993; Hauser et al., 1995). Ten-day-old cob-3 seedlings grown on 4% sucrose medium exhibited short and thick roots and induced the expression of PDF1.2a mRNA, a defence responsive gene, while no changes were detected in the wild-type plants or cob-3 plants grown on 1% sucrose medium (Fig. 7). By contrast, cob-5 showed a ‘constitutive’ phenotype regardless of sucrose concentration. Also, cob-5 constitutively expressed PDF1.2a mRNA, which is consistent with microarray data (Table 1).
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Discussion |
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Complete loss of COBRA function causes severe growth and developmental defects in the whole plant body
Dramatic phenotypic changes are described caused by the null mutation of the COBRA gene, which encodes a glycosylphosphatidylinositol (GPI) anchor cell-surface protein that plays a key role in determining the orientation of cell expansion in roots (Schindelman et al., 2001). It was found that, unlike the previously characterized weak cob alleles, the phenotype of cob null mutants was not restricted to the root tissue but rather was manifested throughout the entire plant body. The most notable aspects of the null phenotype were cell swelling and extreme dwarfism (Figs 1, 2) due to cell elongation defects, and increased pigment accumulation (Fig. 3). Wild-type plants grown for 3 weeks on MS agar medium developed shoots and flowers, whereas cob-5 plants failed to develop shoots and eventually died (data not shown). During the preparation of this paper, Roudier et al. (2005) reported a null-mutant of COBRA (cob-4), which shows identical phenotypes to cob-5. Taken together, these results indicate that the COBRA protein plays an important role in the cellular architecture and growth in both of root and shoot tissues.
Cell expansion defects in Arabidopsis trigger cellular defence responses
Higher plants exhibit remarkable phenotypic plasticity in response to environmental stress. One of the best examples of this phenomenon is induced plant resistance to herbivore attack. During this process, a healthy and rapidly growing plant will activate the massive synthesis of defence-related compounds in response to an inductive cue. Comparison of transcriptional profiles between wild-type and cob-5 plants, together with our biochemical analyses, provide potential clues to the mechanisms by which plant cells initially perceive biotic stress at the cell surface. The most striking feature of the cob-5-induced transcriptome was the massive expression of defence and stress-responsive genes. Most of the up-regulated genes in the cob-5 mutants were also up-regulated in the plants infected by virulent strains of P. syringae (Tables 1, 2; Tao et al., 2003), strongly indicating that defence responses are constitutively activated in cob-5 plants.
Immunolocalization anlaysis indicated that COBRA is targeted to both the plasma membrane and the longitudinal cell walls through a GPI anchoring (Schindelman et al., 2001; Roudier et al., 2005). In addition, cob-4 plants showed disorganization of the orientation of cellulose microfibrils and subsequent reduction of crystalline cellulose in cell wall (Roudier et al., 2005). These findings suggest that COBRA may play a regulatory role in cell wall anisotropic expansion through the organization of cellulose microfibril orientation.
It has been proposed that an alteration in cell wall composition or integrity can mediate signalling events, such as JA- and ethylene-dependent stress and defence responses, in Arabidopsis (Ellis et al., 2002). For example, the recessive cev1 mutant that is defective in a cellulose synthase gene (AtCesA3) exhibits constitutive expression of JA- and ethylene-responsive genes (Ellis and Turner, 2001) and gives enhanced resistance to pests and pathogens. Ellis and Turner (2001) also found that the rsw1 mutant, which is defective in a different cellulose synthase gene (AtCesA1), constitutively expressed the JA-responsive gene VSP. In addition, a mutation of an Arabidopsis chitinase-like gene, which is presumed to be involved in cell wall modification, resulted in over-production of ethylene (Zhong et al., 2002). These results agree with the finding that mutation in a gene regulating cell wall biogenesis activates cellular defences. Furthermore, it has been demonstrated that the induced ‘conditional’ cell elongation defects in cob-3 trigger the expression of a defence-related gene, PDF1.2a (Fig. 7).
GPI modification of proteins occurs in a variety of organisms, including animals, plants, fungi, and protozoa (McConville and Ferguson, 1993; Kinoshita et al., 1997; Takos et al., 1997). The primary role of the GPI anchor is to attach proteins to the outer leaflet of the plasma membrane. In Arabidopsis, 248 proteins with a wide variety of functions are predicted to be GPI-modified, including COBRA (Sherrier et al., 1999; Borner et al., 2002, 2003). Mutations in the pectate lyase-like protein PMR6 alter cell wall structure and pectin content and confer mildew susceptibility (Vogel et al., 2002). Most of the arabinogalactan-proteins (AGPs), which are cell wall proteoglycans, are also predicted to have a GPI modification and have been suggested to be involved in cell-to-cell signalling (Majewska-Sawka and Nothnagel, 2000). Yariv phenylglycoside is a synthetic probe that specifically binds to and aggregates plant AGPs (Yariv et al., 1967; Nothnagel, 1997). Recently, Guan and Nothnagel (2004) reported that treatment of Arabidopsis cell cultures with Yariv phenylglycoside triggered wound-like responses such as cell wall apposition, callose synthesis, and activation of wound-responsive gene expression which was monitored by whole genome array. Indeed, among 411 genes up-regulated in the 1 h treatment of Yariv reagent, 136 genes (>33%) were overlapped with the 2-fold or higher up-regulated genes in cob-5 (see supplementary Table VI at JXB online).
The accumulating evidence suggests that the ability of Arabidopsis cells to sense defects in cell expansion is closely linked to the activation of cellular defence pathways. In the case of cob mutants, loss of COBRA function disturbs anisotropic cell expansion, which in turn leads to the induction of defence signalling as a secondary effect. Further biochemical and genetic analyses are needed to confirm this hypothesis.
New insights gained by whole-genome expression analysis of cob-5 mutants
The defence signalling induced by the loss of COBRA function casts new insight on the cross-talk of JA, SA, and ET pathways. SA has long been known to play a principal role in plant defence against pathogens. Although interaction between the SA and JA signalling pathways are complex, they can be described as being either antagonistic or agonistic to one another (for a review see Kunkel and Brooks, 2002). The pathogen-inducible genes such as PR-1, PR-2, and PR-5 require the presence of normal SA signalling for their transcriptional activation, whereas the plant defensin gene PDF1.2, along with a PR-3 and PR-4 gene, are induced by pathogens via an SA-independent and JA-dependent pathway (Thomma et al., 1998). Interestingly, SA-mediated defence was also significantly manifested in the cob-5 transcriptome since SA biosynthesis-related genes and SA marker genes were also up-regulated (Table 1). Recently, Li et al. (2004) suggested that WRKY70, a member of the WRKY transcription factor family, acts both as an activator of SA-induced genes and a repressor of JA-responsive genes. By comparing our microarray data with that of Li et al. (2004), significant overlap with the cob-5 transcriptome was found (data not shown). Moreover, expression level of WRKY70 increased 3.4-fold in cob-5 plants, suggesting the existence of additional regulatory components between SA and JA.
Many reports provide evidence for positive interactions between the JA and ET signalling pathways (Jackson and Taylor, 1996; Penninckx et al., 1998). However, no significant change was found in the ET-biosynthetic genes in the cob-5 transcriptome (Table 1). Comparative analysis with the published data of Glazebrook et al. (2003) also indicated that the cob-5 transcriptome is highly correlated to the cluster of genes whose expression is dependent on JA signalling but not ethylene signalling (see supplementary Table VII at JXB online). Epistatic analysis between cob-5 and various combinations of pathway-specific mutants would help to dissect the complex defence pathways involving SA, JA, and ET signalling.
In conclusion, it was found that an additional null mutation in the COBRA gene causes a severe defect in a variety of physiological and developmental processes along the whole plant body. Further analysis showed that cellular defence systems were massively up-regulated in cob-5 plants. These observations support the hypothesis that changes in cell wall integrity are intimately associated with the regulation of plant defence responses. Further investigation of cob plants may provide new insight into the mechanism by which cell wall-derived signals co-ordinately regulate cell expansion and responses to biotic stress.
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Supplementary data |
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Supplementary data can be found at JXB online.
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Acknowledgements |
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We thank Merilyn Ruthig for her excellent technical assistance. We are grateful to SIGnAL (Salk Institute Genomic Analysis Laboratory) and ABRC for T-DNA insertional mutant lines. We are also grateful to Dr Annette Thelen and the staff of the Genomics Technology Support Facility (GTSF) at Michigan State University for their help with the Affymerix GeneChip analysis. We thank Dr Shirley Owens in the Center for Advanced Microscopy at Michigan State University for the confocal image analysis. This project is supported by grants from USDA CSREES (No. 98-34158-5995, 00-34158-9236, and 01-34158-11222) to K-HH, and from the National Institutes of Health (R01GM57795) and the US Department of Energy (DE-FG02-91ER20021) to GAH.
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