JXB Advance Access originally published online on May 22, 2007
Journal of Experimental Botany 2007 58(8):2181-2191; doi:10.1093/jxb/erm092
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© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER |
Mutational evidence that the Arabidopsis MAP kinase MPK6 is involved in anther, inflorescence, and embryo development
Genome Center of Wisconsin and Department of Horticulture, 1575 Linden Drive, University of Wisconsin, Madison, WI 53706, USA
* To whom correspondence should be addressed. E-mail: fpat{at}biotech.wisc.edu
Received 20 February 2007; Revised 29 March 2007 Accepted 4 April 2007
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Abstract |
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Loss-of-function, dominant-negative, and change-of-function genetic approaches were used to investigate the role played by the Arabidopsis mitogen-activated protein (MAP) kinase MPK6 throughout development. Plants homozygous for T-DNA null alleles of MPK6 displayed reduced male fertility and abnormal anther development. In addition, a portion of the seed produced by mpk6 plants was found to contain embryos that had burst out of their seed coats. To address potential functional redundancy, a dominant-negative version of MPK6 was constructed by changing the TEY activation loop motif to the amino acid sequence AEF. Plants expressing MPK6AEF via the MPK6 native promoter were found to produce excessive stomata, consistent with the recently described role of MPK6 in stomatal patterning. A novel floral phenotype characterized by abnormal sepal development was also observed in MPK6AEF lines. The gene expression pattern of the MPK6 native promoter was determined using a YFP–MPK6 fusion construct, and expression was observed throughout most plant tissues, consistent with a role for MPK6 in multiple developmental processes. The YFP–MPK6 construct was found to rescue the fertility phenotype of mpk6 null alleles, indicating that the fusion protein retains its biological activity. It was also observed, however, that plants expressing YFP–MPK6 displayed reduced apical dominance and a shortening of inflorescence internodes. These results suggest that the YFP tag modifies the activity of MPK6 in a manner that affects inflorescence development but not anther development. Taken together, the present results indicate that MPK6 is involved in the regulation of multiple aspects of plant development.
Key words: Anther development, Arabidopsis, embryo development, floral development, MAP kinase, MPK6
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Introduction |
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Mitogen-activated protein (MAP) kinase cascades are conserved signalling pathways that regulate diverse aspects of growth, development, and stress response in all eukaryotes. The genome of Arabidopsis encodes 20 MAP kinase isoforms (MAPK-Group, 2002), and much experimental effort has been devoted to determining the biological processes that are regulated by each of these kinases. The particular MAP kinase isoforms MPK3, MPK4, and MPK6 have been the most extensively studied to date. Both biochemical and genetic analyses have been reported for each of these isoforms, and the emerging picture of MPK3, MPK4, and MPK6 signalling is one of complexity (Mishra et al., 2006). Each of these proteins appears to serve in multiple signalling pathways, and at our current level of understanding there seems to be significant functional redundancy between these three proteins. For example, it has been observed that plants exposed to hydrogen peroxide or pathogen-associated molecular patterns (PAMPs) respond by rapidly activating the kinase activities of MPK3, MPK4, and MPK6 (Ichimura et al., 2006; Nakagami et al., 2006; Suarez-Rodriguez et al., 2007). The activation of MPK4 by these signals is dependent on the upstream MAP kinase kinase kinase (MAP3K) MEKK1, whereas activation of MPK3 and MPK6 is not (Ichimura et al., 2006; Nakagami et al., 2006; Suarez-Rodriguez et al., 2007). These results indicate that a given stimulus can activate all three MAP kinases, but via at least two distinct pathways. Genotoxic stress, in contrast, has been shown to activate only MPK6 (Ulm et al., 2002), while ozone treatment activates both MPK3 and MPK6 (Miles et al., 2005). Upstream of the MAP kinases, the MAP kinase kinase (MAP2K) protein MKK1 has been shown to activate MPK4 after PAMP treatment (Meszaros et al., 2006), and after cold and salt stress MKK2 activates MPK4 and MPK6 (Teige et al., 2004). MKK4 and MKK5 activate the kinase activities of MPK3 and MPK6 after PAMP exposure (Asai et al., 2002) and in the stomatal patterning pathway (Wang et al., 2007). These biochemical studies have indicated that a variety of stress treatments result in the activation of different combinations of the three MAP kinase isoforms MPK3, MPK4, and MPK6.
In addition to these biochemical studies, genetic analyses have also been performed for MPK3, MPK4, and MPK6 (Petersen et al., 2000; Menke et al., 2004; Miles et al., 2005; Wang et al., 2007). Null alleles of mpk4 generated by transposon-based mutagenesis have been shown to cause a dwarf plant phenotype due to the constitutive activation of the systemic acquired resistance (SAR) pathway (Petersen et al., 2000; Brodersen et al., 2006). This analysis established MPK4 as a negative regulator of salicylic acid-dependent defence response genes. A deletion null allele of mpk3 isolated by reverse-genetic screening of fast neutron-mutagenized Arabidopsis has been described by Miles et al. (2005) and found to cause an ozone-hypersensitive phenotype. This same study used RNA interference (RNAi) to demonstrate that reduced levels of MPK6 also result in ozone hypersensitivity (Miles et al., 2005). This work indicated that MPK3 and MPK6 may play overlapping roles in oxidative stress response signalling. In a separate study, Menke et al. (2004) also used an RNAi approach to study MPK6 function. These authors observed no abnormal developmental phenotypes in MPK6 RNAi lines but did report compromised resistance to both virulent and avirulent pathogens (Menke et al., 2004). Unlike mpk4 mutants, however, these MPK6 RNAi lines did not affect regulation of the SAR response.
The most recent genetic analysis of MPK3 and MPK6 involved the use of T-DNA null alleles of both loci. In this study, Wang et al. (2007) reported that no obvious developmental phenotypes were apparent in the mpk3 or mpk6 single mutant lines. The double mutant state, however, was found to be embryo lethal. A conditional rescue strategy based on the dexamethasone-inducible promoter was therefore used to overcome the embryo-lethal phenotype and allow observation of the double mutant state later in development. These experiments demonstrated that MPK3 and MPK6 are required for the proper regulation of stomatal patterning in leaf tissue. The absence of MPK3/6 activity causes excessive stomata, while excessive MPK3/6 activity results in an absence of stomata. Additionally, silencing of the MAP2Ks MKK4/5 using tandem RNAi resulted in a stomatal patterning defect similar to that of the mpk3/mpk6 double mutant, demonstrating that MKK4/5 are upstream of MPK3/6 (Wang et al., 2007).
These published genetic experiments, in conjunction with the extensive biochemical investigations that have been reported, indicate that MPK3, MPK4, and MPK6 are involved in the regulation of both stress response and developmental pathways. In the present study a thorough genetic analysis of the MPK6 locus was performed in order to determine if there are additional developmental pathways that make use of MPK6 that have not yet been described. Through the use of loss-of-function, dominant-negative, and change-of-function approaches, several additional aspects of Arabidopsis development that are influenced by MPK6 have been identified. This information serves to broaden our understanding of the many processes that fall under the influence of MAP kinase signalling in Arabidopsis.
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Materials and methods |
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Plant growth conditions and mutants
Arabidopsis plants were grown on soil under constant light at 22 °C. Plants with T-DNA insertions within MPK6 (At2g43790) were obtained from the Salk T-DNA collection (Alonso et al., 2003). SALK_073907 has previously been described as mpk6-2 (Liu and Zhang, 2004); SALK_062471 is denoted here as mpk6-4. The mpk3 mutant used for these studies (SALK_151594) has been described previously (Wang et al., 2007). PCR primers as follows were used for genotyping the plants: MPK6-F2, 5'-GCCTCAGATGCCTGGGATTGAGAATATTC-3'; MPK6-RT-R2, 5'-AGAGTGGCTTACGGTCCATTAACTCCATG-3'; p745, 5'-AACGTCCGCAATGTGTTATTAAGTTGTC-3'; MPK3-F, 5'-CCGAGCAATCTTCTGTTGAACGCGAATTG-3'; and MPK3-R, 5'-TGCTGCACTTCTAACCGTATGTTGGATTG-3'. p745 anneals to the T-DNA left border. Sequencing of the T-DNA flanking sequence for each allele confirmed the identity of the mutated gene and precisely mapped the insert location. All wild-type and mutant plants used in this work are in the Columbia-0 ecotype.
Reverse transcriptase-PCR analysis of gene expression levels
RNA was isolated from plant tissue using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA). cDNA was reverse-transcribed from DNase-treated total RNA using the SuperScript II First Strand cDNA Synthesis System (Invitrogen, Carlsbad, CA, USA). Quantitative real-time PCR analysis was carried out on an iCycler iQTM real-time PCR detection system (Bio-Rad, Hercules, CA, USA) using the following gene-specific primers for MPK6 and the histone gene His2A as a control: MPK6-RT-F2, 5'-GAGGACTCTCCGTGAGATCAAGCTGCTTC-3'; MPK6-RT-R2, 5'-AGAGTGGCTTACGGTCCATTAACTCCATG-3'; H2A-F2, 5'-CGATTTTTGAAAGCCGGTAAGTACGCCGA-3'; and H2A-R2, 5'-GCAACTTGCTTAGCTCCTCATCATTCCTC-3'.
Plasmid constructions
The wild-type MPK6 genomic locus was amplified from wild-type Columbia-0 genomic DNA using the primers MPK6G-F1, 5'-AAAGAAGCTTGGAAAGCAAAGATAAATA-3', and MPK6G-R2, 5'-AAGGAGAGAGAGCTCACAGATGAAAGT-3', and cloned into a plasmid vector. These primers amplify a region containing 
2 kb of promoter sequence, the complete MPK6 coding sequence, and 1 kb of downstream sequence. DNA sequencing was used to confirm that the resulting MPK6 genomic clone did not contain any PCR-induced mutations. For the genetic rescue experiments, this wild-type MPK6 genomic clone was moved into the T-DNA binary vector pCAMBIA3300S (Krysan et al., 2002) and introduced into plants via Agrobacterium-mediated transformation (Clough and Bent, 1998).
The MPK6 amplicon described above was used to create the YFP–MPK6 fusion. Unique AatII and SpeI restriction sites were added directly 3' of the MPK6 start codon using site-directed mutagenesis. The yellow fluorescent protein (YFP)-coding sequence was then PCR amplified and cloned into the MPK6 construct using sticky ends created by AatII and NheI digestion. The resulting N-terminal YFP–MPK6 fusion was moved into the T-DNA binary vector pCAMBIA3300S (Krysan et al., 2002) and introduced into wild-type Columbia-0, mpk6-4, and mpk6-2 plants via Agrobacterium-mediated transformation (Clough and Bent, 1998).
The wild-type MPK6 amplicon was also used to create the dominant-negative MPK6AEF construct. For this construct, the two codons specifying the activation loop residues of MPK6 were modified using site-directed mutagenesis to effect the following changes: T221A and Y223F (Bardwell et al., 1998). The resulting MPK6AEF construct was introduced into Arabidopsis plants as stated above.
Microscopic analysis
To monitor anther development, floral inflorescences of wild-type Columbia-0 and mpk6-2 were fixed in Spurr's resin, dissected into 8–10 mm thin sections, and stained for visualization with 0.05% toluidine blue. Sections were viewed with bright-field illumination.
Environmental scanning electron microscopy (ESEM) was performed using a Quanta 200 ESEM. Fresh cotyledons from 3-d-old wild-type Columbia-0 and mpk6-4+MPKAEF plants grown on 0.5x Murashige and Skoog salt mixture medium with 1% agar (w/v) (MS medium) were placed in the microscope and scanned at 20 kV under 2–4 Torr pressure. The ESEM was also used to examine dehiscent anthers of wild-type and mpk6-2 plants, and developing floral buds of wild-type and MPK6AEF plants.
Three-day-old seedlings expressing YFP–MPK6 were observed using confocal microscopy with a Zeiss Axiovert 100M inverted microscope with Bio-Rad MR1024 laser scanning. A 514 nm laser line from an argon ion laser was used to excite YFP, with the fluorescence emission collected by a broad band-pass filter (480–550 nm). Additionally, an Olympus BX60 compound microscope equipped for epifluorescence analysis of YFP fluorescence was used on cotyledon and floral tissue of plants expressing YFP–MPK6.
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Results |
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mpk6 null alleles cause reduced male fertility due to defects in anther development
Two independent T-DNA null alleles of mpk6 were obtained from the Salk T-DNA collection, and the precise T-DNA insertion sites were determined by DNA sequence analysis (Fig. 1A). The T-DNA insertion in mpk6-2 lies within the fourth exon and that of mpk6-4 is within the third exon. Both of these alleles constitute RNA null mutations based on reverse transcriptase-PCR (RT-PCR) analysis of homozygous individuals (Fig. 1A). Previous reports have indicated that no obvious developmental phenotypes are present in mpk6 mutant plants (Menke et al., 2004; Wang et al., 2007). Under the growth conditions used for the present experiments, however, a significant reduction in the fertility of plants homozygous for either of the mpk6 alleles was observed. In a wild-type Arabidopsis plant, silique elongation is dependent on successful fertilization and subsequent seed development. Silique length therefore constitutes a convenient measure of fertility. As shown in Fig. 1B and C, silique length in mpk6 homozygous plants is
50% less than that of the wild type. Although mpk6 plants have a dramatic reduction in fertility, a small amount of seed is produced by these lines, allowing homozygous individuals to be propagated. Hand pollination of these partially fertile mpk6 plants with wild-type pollen restored the mpk6 siliques to a wild-type size, indicating that a defect in male fertility was responsible for the observed phenotype (data not shown).
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The fact that reduced fertility was observed using two independent alleles of mpk6 provided strong evidence that this phenotype was caused by mutation of the MPK6 locus. In order to confirm this causal role, a genetic rescue experiment was performed by cloning a copy of the wild-type MPK6 genomic locus, moving it into a T-DNA vector, and introducing this construct into mpk6-2 and mpk6-4 plants. This ectopic copy of the MPK6 locus restored full fertility to both the mpk6-2 and mpk6-4 mutant plants, further confirming that mutation of the MPK6 gene is responsible for the reduced male fertility phenotype (data not shown).
One additional aspect of the reduced fertility phenotype that warrants discussion is the fact that this phenotype displays variable penetrance. Under certain growth conditions, progeny from a homozygous mpk6 plant will display a level of fertility that approaches that of the wild type. Under other conditions, however, progeny from that same plant will all display greatly reduced fertility. This variable penetrance may explain why previous studies of mpk6 mutant lines did not report any notable developmental defects. An attempt was made to pinpoint the specific environmental variable that enhanced or suppressed the partial-fertility phenotype by testing different combinations of temperature, humidity, photoperiod, and light intensity. Despite these efforts, the specific environmental factor that tipped the balance between low and high fertility in mpk6 mutant lines could not be determined. It should be noted, however, that even under the most favourable conditions the mpk6 plants never achieve the full fertility of the wild type.
In order to characterize further the nature of the mpk6 reduced male fertility phenotype, mpk6 stigmas were hand pollinated using anthers from the same mpk6 plant. This treatment rescued the reduced fertility phenotype, indicating that mpk6 plants are able to produce at least some quantity of viable pollen grains (data not shown). mpk6 plants therefore seem to be defective in the process of efficiently transferring pollen from anther to stigma. Microscopic analysis was next performed in order to search for possible structural defects that would explain the reduced male fertility. To begin, filament length of the anthers was measured since a common cause of reduced male fertility is a reduction in filament length preventing the anthers from reaching the stigma. No significant difference was observed in the lengths of wild-type, mpk6-2, and mpk6-4 filaments (Fig. 2A; Table 1). ESEM was next used to study anther morphology and it was observed that mpk6 anthers are substantially smaller in size than those of the wild type (Fig. 2B, C). In addition, the individual pollen grains on mpk6 anthers appear to be more tightly associated with the anther when compared with wild type at the same developmental stage, and mpk6 pollen grains may be larger than those of the wild type. Light microscopy of semi-thin sections confirmed that mpk6 anthers were substantially smaller than those of the wild type (Fig. 2D, E). The structural defects observed in mpk6 anthers may be responsible for compromising the efficiency of pollination in these plants, resulting in decreased male fertility.
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mpk6 null alleles cause a defect in embryo development
As part of a comprehensive survey of the development of mpk6 mutant plants, it was observed that a substantial portion of the seed collected from homozygous mpk6-2 and mpk6-4 plants displayed an unusual phenotype in which the embryo was found to be protruding out of the dried seed coat. In order to determine the point in seed development when this abnormality arose, siliques were dissected from plants at various stages and the seeds were observed using light microscopy. As shown in Fig. 3, protruding embryos were detected in mpk6 siliques containing green seeds that had not begun the desiccation stage of seed development. Approximately 7% of the seed produced by mpk6 plants display this protruding embryo phenotype (Table 2). An ectopic copy of the wild-type MPK6 locus provides full rescue of the protruding embryo phenotype (Table 2).
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A survey of the literature indicated that an identical phenotype has been reported for plants carrying a mutation in the YODA gene (Lukowitz et al., 2004), which is a MAP3K that has recently been shown to act upstream of MPK6 and MPK3 to regulate stomatal patterning (Bergmann et al., 2004; Wang et al., 2007). In addition to its role in stomatal development, YODA has also been shown to be required for suspensor development during embryogenesis (Lukowitz et al., 2004). yoda loss-of-function mutants fail to develop a normal suspensor, thereby causing the embryo to become pinned between the walls of the developing seed coat. The resulting physical constraints often cause the embryo to pop out of the seed coat during the course of seed development. The observation of the protruding embryo phenotype in mpk6 plants provides direct genetic evidence that MPK6 acts downstream of YODA in the regulation of embryonic development. Additionally, Wang et al. (2007) demonstrated that mpk3–/– mpk6–/– embryos improperly regulate asymmetric division of the zygote. As a result of these data, and because of the previously described genetic redundancy of MPK3 and MPK6 in the stomatal patterning pathway, seed produced by an mpk3 plant were next analysed, but no protruding embryos were detected (Fig. 3D). The differential effects of mpk3 and mpk6 on embryo development suggest that genetic redundancy between these loci is not complete in the context of embryo development.
A dominant-negative form of MPK6 causes defects in floral development
Loss-of-function alleles represent one genetic strategy for elucidating gene function, but a drawback to this approach is that genetic redundancy can mask the appearance of instructive phenotypes in single mutant lines. In the case of MPK6, it has been previously demonstrated that there is significant redundancy between MPK3 and MPK6 (Wang et al., 2007). It was therefore of interest to determine if alternative genetic approaches would result in the identification of additional development roles for MPK6. Toward this end, a version of the MPK6 gene that encoded a dominant-negative protein was constructed by mutating the coding region such that the canonical TEY activation loop motif was changed to AEF (Bardwell et al., 1998; Petersen et al., 2000). The resulting protein should not be able to be phosphorylated by any of its upstream MAP2K partner(s) such as MKK4/5 (Wang et al., 2007), and therefore should serve to block signalling in its cognate pathways. This MPK6AEF construct uses the MPK6 native promoter to drive transcription.
The MPK6AEF construct was introduced into wild-type Columbia and mpk6 mutant backgrounds, and several independent transgenic lines were isolated for each background. An overproduction of stomata was observed in the leaf epidermis of lines expressing MPK6AEF in either the wild-type or mpk6 mutant backgrounds (Fig. 4D). This result indicated that MPK6AEF is able to overcome the redundancy of MPK3 and block signalling through the YODA–MPK3/6 pathway that regulates stomatal patterning (Bergmann et al., 2004; Wang et al., 2007). RT-PCR analysis was used to measure the expression level of MPK6AEF in three independent transgenic lines. Expression ranged from 2- to 6-fold that of wild-type MPK6 (Fig. 4B). These modest levels of MPK6AEF expression should minimize the likelihood that the MPK6AEF protein interferes with signalling in pathways that are not normally regulated by wild-type MPK6.
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In addition to the stomatal patterning phenotype, a novel floral development phenotype was also observed in MPK6AEF lines (Fig. 5A, B). The most striking aspect of this phenotype is that the sepals of developing flowers are substantially shorter than the carpels and bend outward at the tips. This situation causes the flowers to open at a very early stage in development (Fig. 5C, D). In addition, the overall length of the flowers produced by MPK6AEF plants is substantially shorter than that of the wild type. ESEM analysis indicated that reduced cell elongation in the carpel epidermis was at least partially responsible for this size reduction (Fig. 5E, F). The floral development phenotype was observed in seven independent transgenic lines expressing the MPK6AEF construct in both the wild-type and mpk6 mutant backgrounds.
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MPK6 gene expression pattern
It was observed that MPK6 influences a diverse set of developmental processes. One would therefore predict that MPK6 would be expressed in a variety of tissues in Arabidopsis. In order to test this hypothesis directly, a YFP–MPK6 fusion was constructed and placed under the transcriptional control of the MPK6 native promoter. Transgenic lines were generated by transforming this construct into both wild-type and mpk6 mutant backgrounds. Gene expression was then monitored using epifluorescence and confocal microscopy at various stages of development. As shown in Fig. 6, a strong YFP–MPK6 signal was observed in floral tissues (Fig. 6A, B), leaves (Fig. 6C, D), hypocotyls (Fig. 6E), and roots (Fig. 6F). This broad expression pattern is consistent with MPK6 playing a role in multiple aspects of development.
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The YFP–MPK6 fusion protein causes a novel inflorescence phenotype
When the YFP–MPK6 construct described above was transformed into mpk6 mutant plants it was observed that the partial fertility and protruding embryo phenotypes were both rescued by this fusion protein (Table 2 and data not shown). These results indicated that the YFP tag did not interfere with the biological activity of MPK6. In addition, however, a novel inflorescence phenotype was observed in several independent lines expressing YFP–MPK6 in either the wild-type or mpk6 mutant backgrounds. RT-PCR analysis indicated that these lines expressed YFP–MPK6 at levels ranging from 1–5-fold that of wild-type MPK6 (Fig. 7B).
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The characteristic feature of the YFP–MPK6 inflorescence phenotype is a decrease in apical dominance, accompanied in some cases by a shortening of the internodes between mature flowers (Fig. 7A). In the most drastic examples of this phenotype, the total height of the inflorescence of a mature YFP–MPK6 plant does not exceed 3 cm. For comparison, wild-type plants typically exceed 30 cm in height at maturity. Despite this severe reduction in inflorescence height, the siliques produced by YFP–MPK6 plants are fully fertile and elongate to the size of the wild type. This characteristic indicates that the plants do not suffer from a general reduction in inflorescence growth, but rather have a defect that is specific to internode and stem elongation.
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Discussion |
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Using a combination of genetic approaches, several developmental processes have been identified that appear to be influenced by the Arabidopsis MAP kinase MPK6. Previous reports on the genetic analysis of MPK6 in Arabidopsis have indicated a role for this protein in disease resistance, ozone sensitivity, and stomatal patterning (Menke et al., 2004; Miles et al., 2005; Wang et al., 2007). None of these previous studies reported any reduction in fertility associated with mutation or suppression of MPK6, however, which is contrary to what was observed for the two independent T-DNA null alleles. Since two of these previous studies involved the use of RNAi to reduce the level of MPK6 expression, it is possible that residual levels of MPK6 could explain this discrepancy because the RNAi approach does not always lead to complete gene silencing. It has also been reported, however, that no obvious developmental phenotypes were observed with one of the same T-DNA null alleles of MPK6 that was characterized in the present study (Wang et al., 2007). The likely explanation for this discrepancy is variable penetrance of the male-sterile phenotype caused by mpk6, as discussed in the Results.
It was observed that the partial fertility of mpk6 plants could be overcome by hand pollination using anthers from the same mpk6 plant. This result suggested that mpk6 plants were defective in the movement of pollen from anther to stigma. Microscopic analysis of mpk6 anthers indicated that there was no change in filament length compared with the wild type, but that anther size was significantly reduced in the mutants. The structural abnormalities that were observed in mpk6 anthers may prevent the efficient release of pollen, thereby resulting in partial male sterility.
The other novel phenotype that was observed in mpk6 null mutants was a propensity for the embryos formed on these plants to burst out of their seed coat during the course of seed development. This phenotype was observed in both independent null alleles of mpk6 and was rescued when an ectopic copy of wild-type MPK6 was introduced into the null mutant background. This phenotype is identical to what has been described for plants carrying the yoda mutation (Lukowitz et al., 2004). YODA is a MAP3K that has been shown to regulate cell division in the suspensor during embryo development. YODA loss-of-function mutants have a shortened suspensor that can cause the developing embryo to be squeezed out of the seed coat during the course of seed development (Lukowitz et al., 2004). In addition to its role in suspensor formation, YODA has also been shown to act upstream of MPK3 and MPK6 to control stomatal patterning (Bergmann et al., 2004; Wang et al., 2007). The observation of a yoda-like embryonic phenotype in the mpk6 mutant lines suggests that MPK6 acts downstream of YODA in the regulation of embryo development as well as stomatal patterning.
Because of the functional redundancy that has been documented for MPK3 and MPK6 (Wang et al., 2007), it was of interest to test additional genetic strategies for investigating the function of MPK6. A mutant version of MPK6 was therefore constructed in which the coding sequence for the kinase activation loop is mutated from the native TEY-coding sequence to one encoding AEF. The activation loop of a MAP kinase is a highly conserved motif that is specifically recognized by a cognate MAP kinase kinase (MAP2K) and doubly phosphorylated on the threonine and tyrosine residues (Bardwell et al., 1998). When these threonine and tyrosine residues are replaced with non-phosphorylatable alanine and phenylalanine residues, the resulting kinase should not be able to be activated by MAP2K phosphorylation. The premise for constructing this type of dominant-negative MAP kinase is that the mutant protein should have the capacity to assemble into the native signalling modules normally occupied by wild-type MPK6. Since this mutant form cannot be activated by the upstream MAP2K, however, signalling through this cascade should be blocked. If the MPK6AEF protein is expressed in the mpk6 null mutant background, one would predict that the mutant protein may be able to serve as an effective physical barrier preventing other related MAP kinase isoforms from occupying the void created by the absence of wild-type MPK6 protein. In this way, the MPK6AEF protein could provide a tool for addressing genetic redundancy because it would prevent proteins such as MPK3 from taking over the signalling pathways left vacant in mpk6 null mutants.
One potential drawback to the dominant-negative approach is that the MPK6AEF protein may have the capacity to interfere with MAP kinase cascades that do not normally make use of wild-type MPK6 protein. An attempt was made to minimize this potential complication by putting the MPK6AEF construct under the transcriptional control of the MPK6 native promoter so that the resulting MPK6AEF protein would be present in similar cells and organs, and at levels similar to those of MPK6 in a wild-type plant. When this MPK6AEF construct was transformed into either wild-type or mpk6 backgrounds, a pronounced increase in the number of stomata present in the leaf epidermis was observed, consistent with the mpk3/mpk6 double mutant phenotype described by Wang et al. (2007). The ability of the MPK6AEF construct to interfere with the YODA–MKK4/5–MPK3/6 stomatal development pathway indicates that this dominant-negative approach provides an effective method for overcoming functional redundancy between MPK3 and MPK6.
In addition to the stomatal patterning phenotype which had previously been described by others, a novel floral phenotype was also observed in plants expressing the MPK6AEF construct. The characteristic features of this phenotype were a reduction in sepal and petal size and a pronounced bending of the sepals, resulting in flowers in which the carpel protrudes from the end of the flower very early in development. This novel phenotype indicates that MPK6 plays an important role in signalling pathways that dramatically affect floral organ development. In addition, it is apparent that functional redundancy is masking the appearance of this floral phenotype in mpk6 null mutants, further demonstrating the value of using both dominant-negative and loss-of-function approaches to study gene function.
It has been observed both in the present study and by others that MPK6 is involved in embryo development, leaf development, and floral development (Wang et al., 2007). Correspondingly, expression of a YFP–MPK6 fusion, placed under the transcriptional control of the MPK6 native promoter, indicated that MPK6 is indeed expressed at detectable levels in most tissues of the plant, consistent with its observed functions throughout development. It was also demonstrated that the YFP–MPK6 fusion protein retains its biological activity, as it was able to rescue both the protruding embryo and reduced fertility phenotypes of mpk6 null mutants.
The YFP–MPK6 construct was also found to cause the appearance of a novel inflorescence phenotype characterized by reduced apical dominance and shortened internodes. This phenotype occurred in multiple, independent lines in both the wild-type Columbia and mpk6 mutant backgrounds. In the case of mpk6 plants expressing YFP–MPK6, the decrease in apical dominance was always accompanied by a complete rescue of the reduced fertility phenotype, indicating that the YFP–MPK6 protein is still active in these lines. Based on these results, it appears that the YFP tag is modifying the MPK6 protein in a way that differentially affects its ability to act in particular signalling pathways. It is possible that the signalling pathway affecting internode elongation is composed of different components from those of the pathway that affects anther development, and that the YFP tag interferes with the ability of MPK6 to interact with the internode-specific pathway but not the anther-specific pathway. More detailed structure–function studies using a variety of modified MPK6 fusion proteins could help explain why YFP–MPK6 rescues one mpk6 mutant phenotype while at the same time generating a new mutant phenotype. No matter what the mechanistic explanation is for this novel situation, it serves to highlight the complexity inherent in MAP kinase signalling pathways in Arabidopsis.
It has been demonstrated that the use of a variety of genetic approaches can be an effective strategy for uncovering developmental pathways that utilize a specific MAP kinase in Arabidopsis. The three methods that were employed here (loss-of-function, dominant-negative, and change-of-function) each resulted in the identification of a different aspect of development that was affected by MPK6. MAP kinase signalling pathways may be particularly suited to this type of multifaceted approach because of the extensive complexity and redundancy that exists between these signalling molecules. Continued application of this type of approach to the many members of the MAP kinase gene family should help to improve our understanding of how plants manage and exploit the complexity inherent in MAP kinase signalling pathways.
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Acknowledgements |
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The authors thank Shuqun Zhang for providing seed from the homozygous mpk3 mutant line. This work was supported by a grant from the National Science Foundation (grant number MCB-0447750).
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