Molecular Plant Advance Access originally published online on June 3, 2008
Molecular Plant 2008 1(4):645-658; doi:10.1093/mp/ssn029
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Regulation of Arabidopsis Early Anther Development by the Mitogen-Activated Protein Kinases, MPK3 and MPK6, and the ERECTA and Related Receptor-Like Kinases
a Department of Biology and the Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA 16802, USA
b The Intercollege Graduate Program in Plant Biology, Pennsylvania State University, University Park, PA 16802 USA
c Present address: 3015 Quinby Dr., Columbus, OH 43232, USA
d Present address: 318 Coker Hall (CB# 3280), Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280, USA
e Department of Biology, University of Washington, Seattle, WA 98195, USA
f Present address: Monsanto Company, 700 Chesterfield Pkwy North, Mailzone GG4G, Chesterfield, MO 63017, USA
g Department of Biochemistry and Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA
1 To whom correspondence should be addressed. E-mail hxm16{at}psu.edu, fax 814-863-1357.
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Abstract |
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 TOP Abstract INTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Mitogen-activated protein kinase (MAPK) and leucine-rich repeat receptor-like kinase (LRR-RLK) signaling pathways have been shown to regulate diverse aspects of plant growth and development. In Arabidopsis, proper anther development relies on intercellular communication to coordinate cell proliferation and differentiation. Two closely related genes encoding MAPKs, MPK3 and MPK6, function redundantly in regulating stomatal patterning. Although the mpk6 mutant has reduced fertility, the function of MPK3 and MPK6 in anther development has not been characterized. Similarly, the ERECTA (ER), ERECTA-LIKE1 (ERL1) and ERL2 genes encoding LRR-RLKs function together to direct stomatal cell fate specification and the er-105 erl1-2 erl2-1 triple mutant is sterile. Because the mpk3 mpk6 double null mutant is embryo lethal, anther development was characterized in the viable mpk3/+ mpk6/– and er-105 erl1-2 erl2-1 mutants. We found that both mutant anthers usually fail to form one or more of the four anther lobes, with the er-105 erl1-2 erl2-1 triple mutant exhibiting more severe phenotypes than those of the mpk3/+ mpk6/– mutant. The somatic cell layers of the differentiated mutant lobes appeared larger and more disorganized than that of wild-type. In addition, the er-105 erl1-2 erl2-1 triple mutant has a reduced number of stamens, the majority of which possess completely undifferentiated or under-differentiated anthers. Furthermore, sometimes, the mpk3/+ mpk6/– mutant anthers do not dehisce, and the er-105 erl1-2 erl2-1 anthers were not observed to dehisce. Therefore, our results indicate that both ER/ERL1/ERL2 and MPK3/MPK6 play important roles in normal anther lobe formation and anther cell differentiation. The close functional relationship between these genes in other developmental processes and the similarities in anther developmental phenotypes of the two types of mutants reported here further suggest the possibility that these genes might also function in the same pathway to regulate anther cell division and differentiation.
Received for publication February 25, 2008. Accepted for publication April 5, 2008.
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INTRODUCTION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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The coordination of cell proliferation and differentiation is essential for normal plant growth and fertility. The male reproductive organ, the stamen, is composed of a filament and an anther. The filament provides nutrients and structural support to the anther and elongates prior to and during dehiscence (pollen release). The anther has four lobes where pollen grains are produced. Anther development in Arabidopsis is divided into 14 stages based on anther histogenesis (Sanders et al., 1999). An epidermis encases the entire anther, and, within the locules, adjacent rings of cells are formed surrounding the sporogenous cells. Stage 5 is marked by the establishment of the five cell layers present within each lobe. From outer to inner, they are: epidermis, endothecium, middle layer, tapetum, and pollen mother cells (PMCs). Subsequent to stage 5, PMCs undergo meiosis to produce haploid microspores followed by two mitotic divisions to form pollen. In addition, the tapetum and middle layer degenerate and eventually undergo dehiscence to allow the release of mature pollen grains.
Stamen identity is conferred by the combined action of APETELA3 (AP3), PISTILLATA (PI), and AGAMOUS (AG), which regulate a subset of genes that promote anther development (Bowman et al., 1989, 1991; Ito et al., 2004). The SPOROCYTELESS/NOZZLE (SPL/NZZ) gene encodes a putative transcription factor that acts very early in anther development and promotes microsporogenesis in the anther under the control of AG (Schiefthaler et al., 1999; Yang et al., 1999; Ito et al., 2004). In addition, two other genes encoding CLAVATA1 (CLV1)-related LRR-RLKs, BAM1 and BAM2, act redundantly in anther development to promote the somatic cell types (DeYoung et al., 2006; Hord et al., 2006). Several other genes were found to promote the differentiation of the tapetal cell type, including EXCESS MICROSPOROCYTES1/EXTRA SPOROGENOUS CELLS (EMS1/EXS), SOMATIC EMBRYOGENESIS 1 (SERK1), SERK2, and TAPETUM DETERMINANT1 (TPD1) (Canales et al., 2002; Zhao et al., 2002; Yang et al., 2003; Albrecht et al., 2005; Colcombet et al., 2005; Ma, 2005; Yang et al., 2005).
Normal tapetum development and degeneration are essential for proper pollen development (Mariani et al., 1990, 1991; Denis et al., 1993; Owen and Makaroff, 1995; Wu et al., 1997; Bih et al., 1999; Kapoor et al., 2002; Zheng et al., 2003). Some of the genes known to be important for tapetum development and function in Arabidopsis are MYB33 and MYB65, DYSFUNCTIONAL TAPETUM1 (DYT1), ABORTED MIRCROSPORES (AMS), ARABIDOPSIS THALIANA GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE 1 (AtGPAT1), and MALE STERILE1 (MS1). The myb33 myb65 double mutant and the single mutants of the other genes are aberrant in tapetum development at different stages, but all result in partial or complete male sterility, underscoring the importance of the tapetum in pollen development.
Normal plant growth is dependant upon signal transduction pathways. Signaling pathways have been shown to involve mitogen-activated protein kinases (MAPKs) and/or leucine-rich repeat receptor-like kinases (LRR-RLKs). For example, the MAPKs MPK3 and MPK6 and the LRR-RLKs are involved in pathogen response and are essential for normal guard cell patterning and formation (Yang et al., 2001; Zhang and Klessig, 2001; Asai et al., 2002; Shpak et al., 2005; Wang et al., 2007). Interestingly, several of these signaling components have been shown to play roles in multiple developmental processes. The LRR-RLK SERK1, for example, was originally isolated for its ability to promote somatic embryogenesis (Schmidt et al., 1997; Hecht et al., 2001). In addition to a role in anther development, BAM1 and BAM2 work with BAM3 to maintain meristem size and to promote proper development of leaf vasculature (DeYoung et al., 2006). The ERECTA (ER) gene has long been known to be important for plant height and was recently found to be involved in resistance to bacterial and fungal pathogens (Godiard et al., 2003; Llorente et al., 2005). ER has also been shown to function with ERECTA-LIKE1 (ERL1) and ERL2 to promote cell proliferation, control guard cell differentiation, and direct ovule development (Rédei, 1992; Torii et al., 1996; Shpak et al., 2004, 2005; Pillitteri et al., 2007). MPK3 and MPK6 have also been shown recently to regulate ovule development (Wang et al., 2008). In addition, it has recently been reported that MPK6 and the ER-family LRR-RLKs are involved in stamen development (Shpak et al., 2003, 2004; Bush and Krysan, 2007), although details are not clear.
In this study, we show that MPK3, MPK6, and the ER-family genes are important for cell differentiation during anther development in Arabidopsis. The majority of mpk3/+ mpk6/– mutants and er-105 erl1-2 erl2-1 triple mutant stamens have anthers that do not form several normal cell types. When mutant anthers do form somatic and sporogenous cell types, the cells are larger and more disorganized than wild-type cells. Also, the tapetum and middle-layer cells may be delayed in their development, and the mutant anthers often fail to dehisce. Interestingly, the er-105 erl1-2 erl2-1 triple mutant appears to have a more severe phenotype than the mpk3/+ mpk6/– mutant. Together, these results suggest that MPK3, MPK6, and the ER-family genes are key regulators of cell proliferation and differentiation during anther development.
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RESULTS |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Anther Size and Male Fertility are Reduced in the mpk6-1 and mpk3/+ mpk6/– Mutants
MPK3 and MPK6 are closely related homologs that act redundantly in several biological processes (Yang et al., 2001; Asai et al., 2002; Wang et al., 2007). Together, they are essential for plant development, as the mpk3 mpk6 double homozygous mutant is embryo lethal (Wang et al., 2007). The mpk6 mutant was previously reported to have reduced fertility and smaller anthers compared to wild-type, but a detailed histological analysis has not been completed (Bush and Krysan, 2007). In order to better understand the roles of MPK3 and MPK6 in male fertility, we examined plant growth and anther development in plants carrying a mutation in one or both of these genes (Figure 1). The mpk3 single mutant produced viable offspring and appeared similar to wild-type in overall plant growth and silique length (Figure 1A and 1B and data not shown). In line with previous results, the mpk6-1 mutant displayed both long wild-type-like siliques and shorter siliques and set many seeds (Figure 1C and data not shown) (Bush and Krysan, 2007). The mpk3/+ mpk6-1/– plants did not produce wild-type-like long siliques, indicating that fertility was greatly reduced (Figure 1D). It was found that female fertility was severely impaired (Wang et al., 2008). Furthermore, where pollen grains were clearly visible on the anthers of wild-type, mpk3, and mpk6-1 open flowers, much less or no pollen could be seen on the anthers of mpk3/+ mpk6-1/– flowers (Figure 1E–1H) when plants were grown under long-day conditions (16 h light/8 h dark) and were about half the size of wild-type. When mpk3/+ mpk6-1/– were grown under less stressful conditions, such as moderate light (80–100 m–2 s–1; 12 h light/12 h dark), they were about the same size as mpk6-1/– plants and slightly smaller than wild-type (Wang et al., 2008) and produced less than a quarter of the normal amount of pollen (data not shown).
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To determine whether the mpk3/+ mpk6-1/– and single mutants produce viable pollen, Alexander's staining was performed. While the mpk3 and mpk6-1 mutants produced viable pollen grains, the mpk3 anthers were reduced in size, in both height and width, compared to wild-type (Figure 1I and 1J). The mpk6-1 anther produced less pollen and was smaller in size than both the mpk3 mutant and wild-type anthers (Figure 1I–1K). Furthermore, the mpk3/+ mpk6-1/– mutant anther was even smaller than either single mutant and produced very few pollen grains (Figure 1J–1L). No dead pollen was observed in any of the mutants examined, suggesting that the numbers of meiotic cells were reduced, but either no nonviable pollen was produced or they degenerated relatively quickly. The reduced anther size was further confirmed by scanning electron microscopy (SEM) (Figure 2). Both the mpk3/+ mpk6-1/– and mpk3/+ mpk6-2/– mutants produced anthers that were smaller than wild-type (Figure 2). SEM analysis also showed that the entire mpk3/+ mpk6-1/– flower was smaller overall than wild-type and the pistil was shorter and wider than wild-type.
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The mpk3/+ mpk6-1/– Mutant Anthers are Defective in Cell Differentiation
In order to better understand the developmental defects of mpk3/+ mpk6-1/– anthers, semi-thin transverse sections of wild-type and mutant flowers were prepared and analyzed (Figure 3). At anther stage 2, wild-type anthers are roughly oval-shaped and the clusters of cells at the four corners begin to establish cell layers parallel to the epidermis (Figure 3A). During anther stages 2 and early 3, no significant difference was detected between wild-type and mpk3/+ mpk6-1/– mutant anthers (Figure 3A and 3E and data not shown). At late anther stage 3 to stage 4, wild-type anthers form four distinct lobes and rings of cells became visible in each lobe (Figure 3B). At these stages, the mpk3/+ mpk6-1/– mutant displayed a range of anther phenotypes (Figure 3F). A few mutant anthers produced the four lobes but they appeared smaller than wild-type anthers, while the majority of mutant anthers remain relatively oval-shaped and appear highly vacuolated. Thus, the mpk3/+ mpk6-1/– anther developmental defect could be seen as early as anther stage 4. At anther stages 5 and 6, a transverse section of a wild-type anther is butterfly-shaped and each of the four lobes has four distinct, concentric layers of cells surrounding the pollen mother cells (Figure 3C and 3D). In contrast, a range of defects was observed at these stages in mpk3/+ mpk6-1/– anthers (Figure 3G and 3H). Occasionally, an mpk3/+ mpk6-1/– anther formed all four lobes; however, usually, the mpk3/+ mpk6-1/– anthers appeared misshapen, with one or more lobes failing to form. In general, abaxial lobes appeared to develop more frequently than adaxial lobes. Although cell layers could sometimes be recognized in mpk3/+ mpk6-1/– anthers, often, such layers were not well organized or were otherwise abnormal (Figure 3L). For example, the middle layer was often absent. In addition, presumptive tapetal cells and meiocytes/microspores were sometimes abnormally enlarged and irregularly shaped.
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During stages 7 and 8 in wild-type anthers, microspores are released from tetrads, the tapetum becomes a thin ring of darkly staining cells, endothecium cells are vacuolated, and the middle layer is degenerated (Figure 3I and 3J). At these stages, mpk3/+ mpk6-1/– anther locules occasionally displayed similar cell patterning and structure to wild-type (Figure 3L). However, most of the cell types in mutant anthers appeared abnormal at stages 7 and 8 (Figure 3L and 3M). The mpk3/+ mpk6-1/– mutant tapetum did not form a thin and darkly stained ring around the microspores, but remained thick and disorganized (Figure 3M). Interestingly, unlike mpk3/+ mpk6-1/–, mpk3/– mpk6-1/+ anthers appeared to develop normal cell layers, similar to wild-type (Figure 3N).
The mpk3/+ mpk6-1/– Mutant Inflorescence Displays Reduced Expression of Several Anther Genes
In order to gain insights into the relationship between MPK3/MPK6 and other genes important for anther development, we examined the expression of several genes (Figure 4). In our previous microarray analysis (Wijeratne et al., 2007), the expression levels of MPK3 and MPK6 were not significantly different in the spl or ems1 mutants compared to wild-type, and MPK6 was expressed at a slightly higher level than MPK3 in anthers (Figure 4A). In addition, real-time PCR performed with RNA from wild-type and mutant inflorescences revealed that the expression of several genes important for anther development did not appear altered in mpk3/+ mpk6-1/– flowers (Figure 4B). No significant difference was observed between mutant and wild-type expression of TPD1, EMS1, and MYB33 (Figure 4B). However, the expression of SPL and AMS were significantly reduced in the mpk3/+ mpk6-1/– mutant flowers by about 50 and 75% that of wild-type, respectively (Figure 4B). Interestingly, the mpk6-1 mutant showed an increased expression of TPD1 and MYB33—two genes involved in tapetum development.
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The er-105 erl1-2 erl2-1 Triple Mutant Displays a Range of Anther Defects and Reduced Number of Stamens
As previously described, the er-105 erl1-2 erl2-1 triple mutant forms stamen with anthers that do not differentiate normally (Shpak et al., 2004). To better understand the defect in anther and pollen development, Alexander's stain was used to determine pollen viability in wild-type and mutants (Figure 5). The er-105, erl1-2, erl2-1 single mutants, er-105 erl1-2, er-105 erl2-1, erl1-2 erl2-1 double mutants, and er-105 erl1-2 (+/–) erl2-1 mutant had anthers that were comparable in size to wild-type and produced round, uniform pollen grains that were viable, similar to wild-type (Figure 5A–5H).
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Many of the er-105 erl1-2 erl2-1 flowers did not produce stamens (Shpak et al., 2004 and data not shown). In er-105 erl1-2 erl2-1 flowers that did produce stamens, the anther developmental defect varied in severity. The most severe defect observed was filament-like stamens that appeared to completely lack an anther (Figure 5I). Some stamens had apparently undifferentiated anthers of variable sizes (Figure 5J). A few stamens produced viable pollen in one or two of their anther locules (Figure 5K and 5L). These anthers were larger than those that did not differentiate, but were still drastically reduced in size, mainly in height, but also in width, compared to wild-type.
Interestingly, the average number of anthers in the er-105 erl1-2 erl2-1 triple mutant was reduced and varied more compared to wild-type (3.06 ± 1.18 vs 5.77 ± 0.56, respectively) (Figure 6A). Almost three-fourths of the stamens observed (69/95) had under-developed anthers, about one-tenth were filament-like (10/95), and less than one-fifth (16/95) produced pollen (Figure 6B). No dead pollen was ever observed in er-105 erl1-2 erl2-1 triple mutant anthers. Interestingly, pollen grains in the triple mutant appeared slightly smaller than wild-type (not shown). In addition, although they produced viable pollen, these anthers did not dehisce and pollen grains adhered to the anther wall when anthers were manually broken, indicating additional later defects.
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The er-105 erl1-2 erl2-1 Triple Mutant is Defective in Anther Formation and Differentiation
To gain further insight into the development of anther cell layers, semi-thin transverse sections of wild-type and the ER-family mutants were analyzed (Figure 7 and Supplemental Figure 1). At anther stage 5, each wild-type locule had the five normal cell layers (Figure 7A). Similar to wild-type, the single, double and er-105 erl1-2 (+/–) erl2-1 mutants produced the five anther cell layers (Supplemental Figure 1). In general, these cell layers were similar in appearance to wild-type cell layers.
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Consistent with the er-105 erl1-2 erl2-1 stamen phenotypes observed using the Alexander's stain, cross-sections of the triple mutant flowers showed stamens that were defective in anther development to varying degrees (Figure 7C–7F). Filament-like stamens appeared fairly round with a central vascular bundle (Figure 7E and 7F). Stamens that produced under-developed anthers varied in size and shape, but were overall oval or kidney-shaped (Figure 7C–7F). These anthers generally had a vascular bundle near the center surrounded by large, disorganized cells that were similar in appearance to connective tissue. When anther lobes with the sporophytic cell types were formed, they occupied only the abaxial positions; therefore, no more than two of the four locules were ever observed to form.
In general, when a locule of an er-105 erl1-2 erl2-1 anther was formed, the five cell layers could be seen, on the basis of position and overall appearance. The cells of the epidermis and endothecium appeared slightly larger in the triple mutant than in wild-type, but were otherwise fairly normal. The middle-layer cells appear adjacent to each other in wild-type (Figure 7A and 7B). In contrast, the middle-layer cells appear to overlap with one another in the triple mutant (Figure 7C and 7D). In addition, sometimes, two periclinally adjacent middle-layer cells were observed, indicating an additional cell division, whereas, other times, the middle-layer cells did not appear to completely surround the tapetum (Figure 7D). Furthermore, mutant middle-layer cells were also larger at stage 6 than in wild-type, suggesting that they persisted longer than normal (Figure 7F and 7G). The most striking cellular defect was seen in tapetal cells. In wild-type, the tapetum forms a single layer of rectangular cells surrounding the PMCs at stage 5 (Figure 7A), which become separated from each other and from meiocytes by a callose layer during stage 6 (Figure 7B and 7E). In the er-105 erl1-2 erl2-1 mutant, tapetal cells did not form a single layer, but appeared as a largely disorganized mass of cells that were greater in number compared to wild-type (Figure 7D, 7F, and 7G). In addition, the tapetum did not always completely surround the meiocytes, possibly due to the degradation of some of tapetal cells. Furthermore, the tapetal cells were greatly enlarged and abnormally shaped. They possessed large vacuoles and did not appear to separate from each other during stage 6. When PMCs had developed into microspores (stage 8), tapetal cells remained predominately attached to each other and to other somatic cells surrounding them and their degeneration appeared delayed (not shown).
In summary, the ER-family single, double, and er-105 erl1-2 (+/–) erl2-1 mutants produced viable pollen and formed five normal cell layers similar to wild-type. The er-105 erl1-2 erl2-1 triple mutant produced fewer stamens and was defective in anther development, with phenotypes varying from antherless stamens to anthers with no more than the two abaxial lobes with differentiated cell layers. In addition, cells of the triple mutant anthers appeared larger and more disorganized than wild-type. Tapetum development appeared particularly affected, and tapetum and middle-layer cells may have been delayed in their development. Finally, although the triple mutant anthers sometimes produced viable pollen, they did not dehisce.
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DISCUSSION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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ER/ERL1/ERL2 and MPK3/MPK6 Are Important for Anther Formation and Development
Previous studies indicate that the ER/ERL1/ERL2 and MPK6 genes are involved in stamen development (Shpak et al., 2003, 2004; Bush and Krysan, 2007). Our results demonstrate that the ER/ERL1/ERL2 and MPK3/MPK6 genes are important for normal anther development. Specifically, the ER/ERL1/ERL2 genes function together to regulate both anther lobe formation and anther cell differentiation. The overlapping expression of ER, ERL1, and ERL2 during anther development (Supplemental Figure 2) and normal anther development in the single, double and er-105 erl1-2 (+/–) erl2-1 mutants indicate that these genes act redundantly and that a single functional copy of ERL1 is sufficient for anther development. Interestingly, ERL2 was recently shown to be haploinsufficient for promoting normal ovule development in the absence of ER and ERL1 (Pillitteri et al., 2007). Further study will be required to determine whether ERL2 is also haploinsufficient in directing anther development.
Similarly, our results of the mpk3/+ mpk6-1/– mutant indicate that these two genes together play crucial roles in anther lobe formation and anther cell differentiation. The ability of the mpk3/– mpk6-1/+ mutant to develop anther cell layers, similar to those in wild-type, suggests that one copy of MPK6 is sufficient to direct anther cell differentiation in the absence of MPK3. However, the mpk6-1 mutant sometimes failed to develop the normal cell layers and most of the mpk3/+ mpk6-1/– anthers had an aberrant phenotype (Bush and Krysan, 2007, and this study). Therefore, MPK3 is not sufficient to consistently direct normal cell differentiation in the absence of MPK6. Furthermore, loss of one function copy of MPK3 in the mpk6-1 mutant greatly enhances the mutant phenotype. These observations indicate that the MPK3 and MPK6 genes have partially redundant functions, with MPK6 playing a more prominent role. MPK6 is expressed at a higher level than that of MPK3, providing a possible explanation for the functional difference between these two genes. Because the mpk3 mpk6 double homozygous mutant is embryo lethal, it was not possible to determine the anther phenotypes of the double mutant; nevertheless, it is reasonable to speculate that the double mutant might have more severe anther development phenotypes than the mpk3/+ mpk6-1/– mutant. Because ER/ERL1/ERL2 and MPK3/6 are members of gene families that include more distant members, it is possible other family members may also have some overlapping functions.
The ER/ERL1/ERL2 and MPK3/6 Functions in Anther Development Are Analogous to their Roles in Other Developmental Processes
The ER/ERL1/ERL2 genes promote cell proliferation and control differentiation during plant development (Torii et al., 1996; Shpak et al., 2003, 2004, 2005). For example, the cortex of the er-105 erl1-2 erl2-1 mutant pedicel had somewhat larger, irregularly shaped cells that appeared disorganized and had gaps between them (Shpak et al., 2004). The triple mutant was severely reduced in longitudinal cell number, but not affected in the number of cell files (Shpak et al., 2004). In contrast, the number of guard cells seen in the er-105 erl1-2 erl2-1 mutant epidermis was dramatically increased, indicating a possible synergistic function of these genes in negatively regulating asymmetric cell division and guard mother cell differentiation (Shpak et al., 2005). Thus, the ER-family genes promote cell proliferation and regulate differentiation, linking the two to cell patterning and organ growth (Shpak et al., 2003, 2004, 2005).
Analogous roles can be seen for ER/ERL1/ERL2 in anther development. At very early stages of anther formation and development, ER, ERL1, and ERL2 redundantly promote the propagation of cells necessary for normal anther formation. The enlarged and disorganized cells observed in some of the er-105 erl1-2 erl2-1 triple mutant anthers are similar to the phenotype observed in the pedicel cortex. While disruption of the cell cycle can lead to increased cell size, this does not account for the aberrant cell patterning or increased number of tapetum cells (Urbani et al., 1995; Wang et al., 2000; De Veylder et al., 2001). The ER/ERL1/ERL2 genes may communicate signals necessary for normal tapetum cell division and differentiation, perhaps analogous to their role in stomatal cell specification. In anther locule formation, asymmetric cell divisions occur periclinally, while symmetric cell divisions occur longitudinally and anticlinally. Hence, anther cell differentiation is distinct from epidermal cell patterning in that symmetric cell divisions do not typically occur in the same plane as the asymmetric cell division events (Sanders et al., 1999; Scheres and Benfey, 1999; Geisler et al., 2000; Scott et al., 2004). Therefore, in the er-105 erl1-2 erl2-1 mutant, the lack of ER/ERL1/ERL2 function leads to aberrant cell patterning and increased numbers of tapetum cells and sometimes the middle-layer cells, suggesting a function of these genes in repressing periclinal cell division events and promoting longitudinal cell divisions in anther development, as well as contributing to normal cell patterning.
The phenotypes of the mpk3/+ mpk6/– mutant anthers suggest that the MPK3/6 genes are important for cell division in the very early stages of anther development to promote the formation of anther lobes. In addition, the later mutant defects in the differentiation of anther cell layers, particularly the tapetum, with enlarged cells suggest that the signal transduction process mediated by the MPK3/6 kinases is also important for the regulation of anther cell fates. Both ER/ERL and MPK3/MPK6 are required for proper anther development, and their loss of function led to similar anther phenotypes. The ER/ERL and MPK3/6 genes have also been shown to have similar functions in other developmental processes (Bush and Krysan, 2007; Shpak et al., 2004, 2005; Pillitteri et al., 2007; Wang et al., 2007, 2008). Both the ER/ERL and MPK3/6 genes are key regulators of guard cell patterning (Shpak et al., 2005; Wang et al., 2007). Furthermore, the ER/ERL and MPK3/6 genes are important for ovule development in a haplosufficient manner (Pillitteri et al., 2007; Wang et al., 2008). Our study of anther development further extends the overlapping roles of ER/ERL and MPK3/MPK6 in a broad range of developmental processes.
Possible Relationship between ER/ERL1/ERL2 and MPK3/6 and with Other Anther Genes
ER/ERL1/ERL2 are LRR-RLKs that are thought to mediate signaling events at the cell surface (Torii et al., 1996; Shiu and Bleecker, 2003; Shpak et al., 2003, 2004, 2005; Pillitteri et al., 2007), whereas MPK3/6 are MAP kinases that are components of a signal transduction protein kinase cascade that include MAPKK (MAPK kinase) and MAPKKK (MAPKK kinase) as upstream regulators (Yang et al., 2001). As discussed above, ER/ERL1/ERL2 together are critical for several developmental processes, including cell proliferation, guard cell differentiation and female fertility (Torii et al., 1996; Shiu and Bleecker, 2003; Shpak et al., 2003, 2004, 2005; Pillitteri et al., 2007). In addition, MPK3/MPK6 have also been shown to regulate stomatal and ovule development, and function downstream of MKK4/MKK5, two functionally redundant MAPKKs, and YODA (a MAPKKK) (Bergmann et al., 2004; Wang et al., 2007, 2008). Both the er erl1 erl2 triple mutant and the mpk3/+ mpk6/– mutant show somewhat similar abnormal anther phenotypes, including the failure to form one or more anther lobes and abnormal anther cell differentiation, suggesting that these two sets of genes regulate related aspects of anther development. Therefore, a reasonable hypothesis is that they act in the same pathway that is important for normal anther development.
The observation that both the er erl1 erl2 triple mutant and the mpk3/+ mpk6/– anthers often fail to form one or more anther lobes suggests these genes are important at very early stages of anther development. Detailed analysis of wild-type anther development indicates that archesporial cells are formed in the L2 layer of the anther primordia and they divide to form the four lobes (Sanders et al., 1999). Therefore, the failure to form an anther lobe could be due to either the absence or abnormal function of archesporial cells at one or more locations for future anther lobes, suggesting that the ER/ERL1/ERL2 and MPK3/MPK6 genes either play a role in specifying the archesporial cell identity or in promoting the function of archesporial cells to generate the proper progeny cells. Previous studies indicate that the SPL and BAM1/2 genes are important for controlling the cell differentiation of archesporial cell progeny (Yang et al., 1999; Hord et al., 2006). In the spl and bam1 bam2 anthers, cell differentiation is dramatically altered, although a four-lobed structure can still be formed. In particular, the spl mutant fails to form L2 derived somatic cell types and sporogenous cells. We found that the SPL expression was approximately half of the normal level in the mpk3+/– mpk6/– mutant, whereas the MPK3 and MPK6 expression did not seem affected in the spl mutant. It is possible that ER/ERL and MPK3/6 modulate the expression of SPL, although these genes are not required for SPL expression, consistent with the formation of some lobes in er erl1 erl2 and mpk3/6 mutants.
Among the anther cell differentiation defects we observed in this study, tapetal cells were disorganized with abnormally large vacuoles. Recently, it has been shown that the ems1/exs, serk1 serk2, and tpd1 mutants fail to produce the tapetum layer and form excess PMCs instead, indicating that these genes likely function in the same pathway to specify tapetum formation (Canales et al., 2002; Zhao et al., 2002; Yang et al., 2003; Albrecht et al., 2005; Colcombet et al., 2005). As the er-105 erl1-2 erl2-1 and mpk3/+ mpk6/– anthers can still form the tapetum, the ER/ERL and MPK3/6 genes probably act in a pathway distinct from that involving the EMS1/EXS, SERK1, SERK2, and TPD1 genes. This is further supported by the observation that MPK3/6 were expressed at near normal levels in the ems1 mutant and EMS1 and TPD1 expression was not reduced in the mpk3/+ mpk6/– mutant. In the myb33 myb65 and dyt1 mutants, the tapetum becomes highly vacuolated around anther stage 5 (Millar and Gubler, 2005; Zhang et al., 2006). Subsequently, the tapetal cells enlarge inward, leaving little space inside the locule, while the PMCs degenerate (Millar and Gubler, 2005; Zhang et al., 2006). However, neither of these phenotypes was observed in the er-105 erl1-2 erl2-1 mutant. Furthermore, none of the known loss-of-function Arabidopsis tapetum mutants resembles the er-105 erl1-2 erl2-1 mutant in abnormal tapetum patterning (Wilson et al., 2001; Canales et al., 2002; Ito and Shinozaki, 2002; Sorensen et al., 2002; Zhao et al., 2002; Steiner-Lange et al., 2003; Yang et al., 2003; Zheng et al., 2003; Albrecht et al., 2005; Colcombet et al., 2005; Millar and Gubler, 2005). Therefore, the ER/ERL1/ERL2 and MPK3/6 genes define a new function in the regulation of anther development.
Implication of the Variable Phenotypes in er erl1 erl2 and mpk3/+ mpk6/– mutants
One striking feature shared by both the er erl1 erl2 and mpk3/+ mpk6/– mutants is that the anther phenotypes are quite variable, ranging from anthers lacking any lobes to anthers with two fully formed lobes containing several cell types, unlike previously described mutants, such as spl, bam1 bam2, or ems1, which produce anthers that have rather consistent morphologies. As discussed above, the lack of some anther lobes is likely due to a defect in the formation or function of archesporial cells. However, the fact that some lobes are formed indicates that these genes are not essential for archesporial cell formation or function. It is possible that mutants still had some residual function due to the existence of one functional copy (MPK3) or even potential functionally overlapping paralogs in these families; such partial function might be near the threshold of the needed activity, and small fluctuations of gene function might be just enough for archesporial formation or function sometimes, but not other times.
Alternatively, the function(s) provided by the ER/ERL and MPK3/6 genes might not be essential but serve to modulate the activity of other essential genes, such as SPL. In this scenario, the ER/ERL-mediated signaling event and MPK3/6-dependent signal transduction promote archesporial formation or function by enhancing the expression of SPL and/or other key anther developmental genes. In particular, because the adaxial lobes normally develop slightly later than the abaxial ones and, in the mutant anthers, the adaxial lobes were more likely to be absent, perhaps ER/ERL and MPK mediate cell–cell communications that assist the formation of the adaxial lobes following the initiation of the abaxial lobes. In other words, the ER/ERL and MPK3/6 genes may increase the robustness of the regulatory machinery of early anther development, thereby ensuring proper cell division and differentiation. In the absence of the ER/ERL and MPK3/6 gene functions, the genes that directly control anther development, such as SPL, are vulnerable to factors that disrupt the normal activity of these genes. The fact that the ER/ERL and MPK genes likely mediate cell–cell communication suggests that proper coordination between cells is important for robust anther development. Such a mechanism of coordination might extend to other developmental processes, given that the ER/ERL and MPK3/6 genes are also required for stomatal patterning and ovule development (Bush and Krysan, 2007; Shpak et al., 2004, 2005; Pillitteri et al., 2007; Wang et al., 2007, 2008).
A third possibility is that these genes are important for promoting anther development under variable environments, since ER and MPK3/6 are known to be environmentally responsive (Zhang and Klessig, 2001; Godiard et al., 2003; Llorente et al., 2005; Wang et al., 2007). It is possible that slightly different growth conditions can impact normal anther development; in wild-type, genes such as ER/ERL and MPK3/6 act to modulate the intrinsic anther developmental program to allow proper development in response to environmental changes. When these genes are mutated, response to environmental changes is hampered, resulting in developmental defects. Depending on the environment, the defects might be severe or mild. This type of speculation is supported by the observation that the mpk3/+ mpk6/– mutant can have milder phenotypes under some growth conditions, such as reduced light intensity and/or duration (data not shown). Because plants naturally experience variable growth conditions, genes that promote normal development under environmental stresses likely play important roles in ensuring developmental successes. The variable cellular defects in anther development of er/erl and mpk3/mpk6 mutants revealed by this study provide potentially valuable tools to unravel the RLK-MAP kinase signal transduction pathways that might be crucial for plant development in response to environmental changes.
It is known that the number of pollen-producing sacs (anther lobes in Arabidopsis) in a stamen can vary among related species. For example, species of Chloranthus (Chloranthaceae) have evolved to have twice as many pollen-producing sacs as species of Sarcandra, the closest relative of Chloranthus (Kong et al., 2002). In addition, when microsporogenesis was induced in petal-like organs of the Arabidopsis agamous mutant by AGAMOUS-independent SPL expression (Ito et al., 2004), often, two locules are formed, suggesting that two locules might be a more basal state. Because the er erl1 erl2 triple mutant and mpk3+/– mpk6-/– anthers produced reduced number of locules, it is tempting to speculate that these genes and their counterparts in other plants might have also contributed to the evolution of the number of pollen sacs during angiosperm history.
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Plant Material and Growth Conditions
The mpk6-1 (CS31099), mpk6-2 (Salk_073907), and mpk3 (SALK_151594) T-DNA insertion mutants have been previously described (Liu and Zhang, 2004; Wang et al., 2007). The mpk6-1 mutant was originally in the Ws-0 background and obtained from the Wisconsin Arabidopsis Knockout Facility, and the mpk6-2 mutant, Col background, was obtained from the Arabidopsis Biological Resource Center (ABRC). The mutant alleles used for ERECTA (At2g26330), ERECTA-LIKE1 (ERL1) (At5g62230), and ERL2 (At5g07180) were described previously (Torii et al., 1996; Shpak et al., 2004). Briefly, the er-105 allele was generated in the Columbia (Col) ecotype in the glabrous1 (gl1) mutant background by fast-neutron-irradiated seeds and contains a T-DNA insertion from an unknown source in the ERECTA promoter region (Lehle Seeds, Round Rock, TX, USA) (Torii et al., 1996). The erl1-2 and erl2-1 mutants carry T-DNA insertions in the LRR domain and were generated by the ABRC (Shpak et al., 2004). Col plants were used as the wild-type control and gl1 mutant as a background control for the er-105 mutant. Arabidopsis thaliana seeds were planted directly, or transplanted after germinating on MS plates, on potting mixture and were grown with a 16 h light/8 h dark cycle at 18–23°C.
Expression of ERECTA, ERL1 and ERL2 in Arabidopsis Anthers
The GUS expressing lines used were described previously (Shpak et al., 2004). GUS staining of floral tissue was performed essentially as described previously (http://www.its.caltech.edu/
plantlab), placing the tissue directly into 100% ethanol after staining instead of going through a graded ethanol series. Anthers and other floral organs were dissected and images were taken using a Nikon dissecting microscope (Nikon Corp., Tokyo, Japan) and an Optronics Digital camera (Optronics Inc., Goleta, CA, USA).
Characterization of the Mutant Phenotype
Pollen viability was determined by fixing flowers in Carnoy's fixative (100% ethanol:Chloroform:Acetic Acid = 6:3:1) for at least 1 h and staining with Alexander's stain for at least 7 h at 55–73°C (Alexander, 1969). Flowers were then briefly washed with 10% glycerol and anthers were dissected from them. Images were taken using either a Nikon Eclipse E400 or E800 microscope (Nikon Corp.) with an Optronics Digital camera (Optronics Inc). Average anther number, standard deviations and percent anther type were calculated using Microsoft Excel (Seattle, WA, USA). Flower buds and inflorescences were prepared for sectioning by embedding in Spurr's resin as previously described (Owen and Makaroff, 1995; Zhao et al., 2002). Semi-thin (0.5 µm) sections were made using an Ultracut UCT ultramicrotome (Leica Microsystems, Wetzlar, Germany) and were stained with either 0.05 or 0.1% of Toluidine Blue in 0.05 or 0.1% Na2B4O7 (respectively) for up to 1 min. Images were photographed using an Olympus BX51 microscope (Tokyo, Japan) and a SPOT II RT Slider digital camera with SPOT software version 3.5.8 for Windows (Diagnostic Instruments, Inc., Sterling Heights, MI, USA). The SEM experiment was performed as described previously (Hu and Ma, 2006). Images were edited using PHOTOSHOP 7.0 (Adobe system Inc., San Jose, CA, USA).
Expression Analysis
A set of genes essential for anther development were selected for expression analysis by real-time PCR. The primers for real-time PCR are listed in Supplemental Table 1. Inflorescences at approximately floral stages 1 through 10 were collected and immediately frozen in liquid nitrogen. The total RNA extraction, cDNA synthesis, real-time PCR, and data analysis were performed as described previously (Ni et al., 2004; Zhang et al., 2006).
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Supplementary Data are available at www.mplant.oxfordjournals.org.
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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This work was supported by grants from the Department of Energy (DE-FG02-02ER15332) to H.M., from the Department of Energy (DE-FG02-03ER15448) to K.U.T., and from the National Science Foundation (IBN-0133220) to S.Z.
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Acknowledgements |
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We would like to thank Danielle Asquino for assistance in quantifying the mutant phenotype. We also express appreciation to Bridget Leyland, Gavilange Nestor, Danielle Asquino, and Jiong Wang for help with plant care, and Yi Hu for help with photography of GUS-stained flowers.
No conflict of interest declared.
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2 These authors contributed equally to this paper.
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