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First published online July 31, 2009; 10.1105/tpc.108.065284 The Plant Cell 21:1957-1971 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
Partitioning the Apical Domain of the Arabidopsis Embryo Requires the BOBBER1 NudC Domain Protein[W],[OA]
a Department of Genetics, University of Wisconsin, Madison, Wisconsin 53706 1 Address correspondence to kbarton{at}stanford.edu.
The apical domain of the embryo is partitioned into distinct regions that will give rise to the cotyledons and the shoot apical meristem. In this article, we describe a novel screen to identify Arabidopsis thaliana embryo arrest mutants that are defective in this partitioning, and we describe the phenotype of one such mutant, bobber1. bobber1 mutants arrest at the globular stage of development, they express the meristem-specific SHOOTMERISTEMLESS gene throughout the top half of the embryo, and they fail to express the AINTEGUMENTA transcript normally found in cotyledons. Thus, BOBBER1 is required to limit the extent of the meristem domain and/or to promote the development of the cotyledon domains. Based on expression of early markers for apical development, bobber1 mutants differentiate protodermis and undergo normal early apical development. Consistent with a role for auxin in cotyledon development, BOBBER1 mutants fail to express localized maxima of the DR5:green fluorescent protein reporter. BOBBER1 encodes a protein with homology to the Aspergillus nidulans protein NUDC that has similarity to protein chaperones, indicating a possible role for BOBBER1 in synthesis or transport of proteins involved in patterning the Arabidopsis embryo.
In angiosperms, the apical half of the globular embryo gives rise to the shoot apical meristem (SAM) and the cotyledon(s). In Arabidopsis thaliana, as in other dicots, the SAM develops between the two cotyledon primordia. This is readily apparent at the heart stage of embryogenesis when the two cotyledons (lobes of the heart) have emerged, flanking the developing SAM. The cotyledon primordia, or seed leaves, are determinate, leaf-like organs with limited growth potential. The SAM, by contrast, has much more extended growth potential. Stem cells within the SAM give rise to the majority of the aboveground portion of the plant. Genetic and molecular analyses have identified two types of genes involved in development of the apical half of the embryo. The first type includes those genes that are required for the correct development of the cotyledons or the meristem but do not affect the placement or number of either. The second type includes those genes involved in partitioning the SAM into cotyledon and SAM domains.
Examples of the first type are the Arabidopsis WUSCHEL (WUS), CUP-SHAPED COTYLEDON1-3 (CUC1-3), and SHOOTMERISTEMLESS (STM) genes (Barton and Poethig, 1993
The ALTERED MERISTEM PROGRAM (AMP) and PINOID (PID) loci are examples of the second type of gene, one that alters partitioning of the apical domain of the embryo. amp1 and pid mutants typically make three or more cotyledons (Chaudhury et al., 1993
Several additional observations have implicated the plant growth hormone auxin in the specification of cotyledon primordia (reviewed in Jenik and Barton, 2005
In contrast with amp1 and pid mutants, which make too many cotyledons, topless (tpl) mutant seedlings fail to make both cotyledons and the SAM (Long et al., 2002
Recently, two receptor-like kinase genes have also been implicated in cotyledon formation and apical partitioning. When both the RECEPTOR-LIKE PROTEIN KINASE1 (RPK1) and TOADSTOOL2 (TOAD2) genes are mutant, embryos lack cotyledons, fail to express the ANT1 cotyledon marker, and show expanded expression of the STM meristem marker (Nodine and Tax, 2008
To date, mutants identified in patterning the apical domain of the embryo have been isolated in screens performed at the seedling stage. There are at least two reasons that more mutants have not been found. First, there is likely extensive genetic redundancy as is shown in the case of the rpk1 toad2 mutants (Nodine et al., 2007
Screen for Embryo Arrest Mutants That Misexpress a Reporter for the STM Gene With the goal of isolating embryo lethal mutants that are defective in partitioning the apical domain of the embryo, we mutagenized a line carrying a reporter for the STM locus. This reporter expresses the β-glucuronidase (GUS) enzyme marker in the developing SAM (Figure 1 ; McConnell and Barton, 1998  ; Jenik et al., 2005). The reporter is not expressed throughout the SAM but is excluded from the central region of the developing meristem (Figure 1D).
Following ethyl methanesulfonate mutagenesis, M2 seeds were harvested from individual M1 plants and planted to soil. A total of 1695 independent embryo arrest mutants, occurring in 819 M2 families, were identified. (Embryo arrest mutants found in the same M2 family were considered independent if individual M2 plants segregated embryos arrested at distinct stages.) Arrested embryos developing in the siliques of M2 plants were dissected out of the ovules when siblings were at heart stage and stained with GUS staining solution. In  15% of siliques segregating embryo arrests, neither the wild-type nor the arrested embryos showed GUS expression, presumably reflecting silencing of the transgene. Among those siliques where the transgene remained active, 69 embryo arrest mutants (4% of total) were found to have ectopic expression of STM-GUS, while 182 embryo arrest mutants (11% of total) failed to express the STM-GUS reporter. In all cases, the wild-type siblings of the arrested embryos expressed STM-GUS in the wild-type fashion. Mutant embryos ectopically expressing STM showed a range of phenotypes with regard both to stage of arrest and to pattern of ectopic expression. Most ectopically expressing mutants formed cotyledons flanking a broader domain of STM expression. A few mutants showed ectopic expression and arrested at the globular stage; examples of such mutant embryos are shown in Figure 1. One type of mutant (represented by one mutation, the bobber1 mutation described below) expressed the STM reporter throughout the top half of the globular embryo (Figure 1G). A second type of mutant (represented by two mutations) expressed the STM reporter throughout the globular embryo (Figure 1H). A third type of mutant (represented by one mutation) expressed the STM reporter in a hypertrophied suspensor (Figure 1I). Of the mutants that failed to express the STM-GUS reporter, most (154) arrested at the globular stage (Figure 1K). A minority of the nonexpressing class of embryo arrest mutants (26) developed past the globular stage (Figure 1J). Because STM reporter expression does not begin in the wild type until the late globular or transition stage, a globular arrest mutant that fails to express the reporter may do so because it is blocked in its development and has not matured to the stage at which STM is expressed. Alternatively, it may do so due to a patterning defect, such as failure to specify a SAM domain. Because we cannot distinguish between these two possibilities, we chose to focus on an ectopically expressing line for in-depth study rather than a nonexpressing line.
The bob1 Mutant Arrests at the Globular Stage and Shows Ectopic STM Expression
The initial assay for STM:GUS reporter expression in a bob1 mutant embryo revealed that the expression of STM, normally confined to a few cells in the middle of the apical portion of the embryo, is expressed throughout the apical half of bob1 embryos (Figure 1G). STM expression begins in wild-type embryos at the late globular or transition stage. By the time wild-type siblings have reached early heart stage, bob1 mutant embryos have begun to express the STM:GUS reporter. In situ hybridization experiments on siliques from plants heterozygous for the bob1 mutation using an antisense STM probe confirm that STM is ectopically expressed in bob1 embryos (Figures 2F and 2G). Expression in the entire apical hemisphere of the globular embryo indicates that STM is expressed in cells that would normally be specified as cotyledon tissue.
bob mutant embryos can reliably be distinguished from their wild-type siblings beginning when the wild-type embryos are at heart stage. In siliques of bob1/+ plants, one-quarter of the embryos remain at early globular stages, while none do so in siliques on +/+ sibling plants. (A total of 15/50 embryos in bob1/+ siliques were at the 16-cell stage or earlier; the remainder were beyond the 32-cell stage, with the majority [22] being at the transition stage. For +/+ siliques, 0/43 embryos were at the 16-cell stage or earlier, with the majority [20] being at the transition stage.) The presumed homozygous bob1 embryos have normal division patterns at the 8- and 16-cell stage (Figures 2C and 2D; see also sections of bob embryos in Figures 3 to 5
Although bob1 mutant embryos arrest at the globular stage, they do not die at this point. Experiments that rely on the continued ability of bob1 embryos to maintain undegraded RNA (in situ hybridizations) and protein (STM:GUS reporter protein assays) indicate that they remain viable until the time that the wild-type embryos in the silique mature and desiccate. To test if bob1 embryos would recover and restart their development if given enough time, we placed arrested bob1 embryos on three different types of tissue culture media and evaluated their reactions (Murashige and Skoog, 1962 ; Fischer and Neuhaus, 1996; Li and Thomas, 1998). bob1 embryos were dissected from their seed coats when wild-type siblings were at the bent-cot stage. Although the wild-type siblings placed on Fisher and Li media grew and became wild-type-looking seedlings, none of the bob1 embryos on any of the media ever grew or developed any further.
bob1 Embryos Are Not Rescued by Removing STM Function
The self-progeny of 16 F1 progeny from four different bob1/+ by stm-11/+ crosses were examined and the number of bob1 and wild-type embryos recorded (see Supplemental Table 1 online). bob1 individuals accounted for 23.5% of the embryos in the siliques of all of the bob1/+ stm-11/+ individuals combined.
Expression of Markers for Apical Development in bob1 Embryos
Our results indicate that CUC2 is first expressed in the entirety of the apical region of the wild-type 16-cell stage embryo (Figure 3A). This is earlier and more extensive than has been reported previously (Aida et al., 1999 In bob1 mutant embryos, CUC2 expression is initially similar to that seen in wild-type embryos; it accumulates throughout the apical domain of the 16-cell stage embryo and then becomes limited to the subepidermal cells of the late globular stage embryo (Figure 3E). However, further limiting of CUC2 to a central stripe separating two cotyledon domains does not appear to occur, and the CUC2 domain of expression may be slightly expanded relative to that of the wild type (cf. Figures 3F and 3G to 3D). In summary, expression of CUC2 is expanded in bob1 mutant embryos but is less affected than the expression of STM.
The exclusion of CUC2 from the epidermis in both wild-type and bob1 mutant embryos indicates that the specification and early development of the protoderm is normal in bob1 mutant embryos. To further test this, we examined the expression of the ARABIDOPSIS THALIANA MERISTEM LAYER L1 (ATML1) mRNA in wild-type and bob1 mutant embryos. The ATML1 gene encodes a transcription factor in the HDZIPIV class (Lu et al., 1996
The expanded domain of expression of STM suggests that bob1 embryos are defective in specification of the cotyledon primordia. To test this, we examined expression of the ANT1 mRNA in wild-type and bob1 mutant embryos. The ANT1 gene is expressed in all the lateral organs of the plant, including the cotyledons in the embryo (Elliott et al., 1996
Like STM and CUC2, the WUS gene is required for the normal establishment of the SAM during embryogenesis. However, WUS is expressed in a pattern that is distinct from that of either STM or CUC2. In wild-type embryos, the WUS transcript accumulates from the 16-cell stage throughout embryogenesis, first appearing in all nonprotoderm cells of the apical embryo, then in a progressively smaller region as cell divisions narrow its pattern to a final expression domain that is one to two cells wide and one cell deep in the L3 layer of the SAM in the mature embryo (Mayer et al., 1998 Accumulation of low levels of the WUS transcript can be seen in bob1 embryos in variable positions. A small region of expression is typically seen somewhere in the apical domain of the embryo (Figure 3J). This region is often off center. It appears that the mRNA accumulates in a slightly different position in each embryo, as if the positional cues WUS needs for correct localization are disrupted or destabilized. However, unlike STM, the number of cells expressing WUS is not increased.
UNUSUAL FLORAL ORGANS (UFO) appears in a subset of meristem cells, beginning around the early heart stage of embryogenesis. Its expression in the embryo is confined to the cells of the presumptive SAM. By the torpedo stage, expression is narrowed to a cup-shaped subdomain within the STM expressing domain (Long and Barton, 1998 UFO is expressed in bob1 mutants, but in a manner that suggests that the positional cues UFO needs for proper expression are disrupted (Figure 5 ). The intensity and area of expression are variable from embryo to embryo. Expression is largely, if not entirely, confined to the protoderm in the apical portion of the embryo. Thus, as in the wild type, expression of UFO is limited to a subdomain of STM expressing cells, which is consistent with a requirement for STM plus another factor in activating UFO expression.
Recently, directed transport of auxin into the cotyledon domains has been proposed as a mechanism to explain the early events in specification of cotyledon primordia (for review, see Jenik and Barton, 2005
Auxin maxima in the embryo have been associated both with the developing cotyledon primordia as well as with the developing root pole of the embryo. To determine if gene expression patterns are abnormal at the root end of the embryo, we analyzed expression of the SCARECROW gene. SCARECROW expression is first seen at the globular stage, where it is found in the lenticular cell (Figure 7 ). Expression expands to include the endodermis as the embryo progresses through the heart and torpedo stages. By contrast, bob mutant embryos fail to express the SCARECROW transgene. Thus, gene expression associated with the auxin maximum at the root pole of the embryo is also disrupted in bob mutant embryos.
BOB1 Encodes a Gene with Similarity to the Aspergillus nidulans NUDC Gene The 53-kb BOB1 region contained 10 predicted genes (see Supplemental Figure 1 online). Predicted genes were amplified from wild-type Landsberg erecta (Ler) genomic DNA and their sequences compared with published sequences from the same open reading frames in wild-type Columbia (Col). Polymorphic loci in the coding sequences of the two ecotypes were identified. Predicted genes were then amplified out of genomic DNA from an F1 plant resulting from a cross between a bob1/+ plant and a wild-type Col plant. Because the bob1 mutation is on a Ler chromosome, the F1 plant contains one Ler chromosome containing the mutation and one wild-type Col chromosome. Amplification reaction products should be 50% from the Ler allele and 50% from the Col allele. Amplification products were cloned into plasmid vectors, and the inserts of several independent clones were sequenced and examined for the presence of a mutation. To ensure that both chromosomes present in the bob1/+ F1 plant had been examined for mutations, the sequences were compared in amplification products containing polymorphisms between Ler and Col sequences.
Sequencing of the MYN8.1 locus (At5g53400) from the Ler chromosome of a bob1-1/+ plant revealed that it contained a C-to-T transition that created a stop codon at amino acid position 196 of the predicted protein (see Supplemental Figure 1 online; Figure 8
). To ensure that this was the mutation causing the bob1 phenotype, a 6-kb fragment of MYN8 DNA was amplified and put into binary vector pCAMBIA 1300 for transformation into wild-type Arabidopsis (see Supplemental Figure 2 online). The rescue plasmid was named pREJ14. After transformation, wild-type Ler plants resistant to hygromycin were crossed to bob1/+ plants. The hygromycin-resistant F1 progeny from this cross were allowed to self-fertilize, and the embryos inside self-crossed siliques were examined for segregation of bob1 embryos. bob1 and wild-type embryos in siliques from F1 plants that segregated bob1 embryos were counted (see Supplemental Table 3 online). A total of 5.6% of these embryos displayed the bob1 phenotype, which is in agreement with 1/16 bob1 embryos, the expected ratio for self-progeny segregating from a parent heterozygous for the bob1 mutation and hemizygous for a rescuing plasmid (
Finally, a second allele (bob1-2) containing a T-DNA insertion in the N terminus of bob1 at Ile-117 (Salk_001125) was obtained from the Salk T-DNA collection. Self-pollinated bob1-2 siliques contain embryos arrested at the globular stage in a 1:3 ratio. To show that these aborted embryos were due to a lesion in BOB1, crosses between bob1-1 and bob1-2 heterozygous plants were performed. bob1-1 failed to complement the bob1-2 aborted embryo phenotype (see Supplemental Table 4 online). These genetic and molecular complementation experiments demonstrate that the bob1-1 embryo arrest phenotype is due to mutations in the MYN8.1 (At5g53400) gene. To demonstrate that the STM mislocalization phenotype observed in bob1-1 arrested embryos is due to the bob1-1 lesion as opposed to a separate linked mutation, we further characterized bob1-2. Plants heterozygous for bob1-2 were crossed to the STM:GUS reporter. Arrested embryos in the siliques of the self-pollinated progeny of this cross displayed STM expression, which was similar to the mislocalization observed in bob1-1 (see Supplemental Figure 3 online). Since two independent bob1 alleles both display STM mislocalization, we conclude that this phenotype is specifically due to lesions in BOB1.
BOB1 encodes a homolog of the A. nidulans NUDC (for Nuclear Distribution C) gene, and NudC domain containing proteins are found in fungi, animals, and plants (Figure 8). There is one close BOB1 homolog in the Arabidopsis genome that is contained in a BOB1 syntenous region (Freeling et al., 2007
BOB1 Is Expressed throughout the Early Arabidopsis Embryo
A BOB1:GUS reporter transgene was constructed and named pREJ19. BOB1 coding sequences with introns and
The same promoter fragment was used to drive expression of a BOB-GFP fusion protein. A genomic fragment including the promoter plus BOB exons and introns was fused to GFP. This construct was capable of rescuing bob null mutants. Analysis of embryos expressing this construct shows that the BOB protein accumulates throughout the cytoplasm (Figure 9
). In heart stage embryos, the protein may be somewhat enriched in epidermal cells. The location of the BOB protein in the cytoplasm is consistent with a predicted role of NUDC-like proteins as chaperones (Faircloth et al., 2009
BOB1 Is Required for Establishment of Normal Patterning in the Apical Domain of Arabidopsis Embryos Early in dicot embryo development, the top half of the globular embryo becomes partitioned into domains that will form the cotyledons and the SAM. To identify genes required for this partitioning, we designed a mutant screen to isolate mutants with altered expression of a meristem-specific marker, the STM:GUS reporter gene. We limited our screen to mutants that arrest as embryos. This was in part because extensive screens for pattern defective mutants have already been performed at the seedling stage. It was also because it is possible that severe disruption of early patterning events may lead to embryo arrest. Our screen identified one mutant, the bob1 mutant, in which the STM:GUS reporter is expressed throughout the top half of the globular embryo extending into the regions that would normally form the cotyledons. Moreover, bob1 mutants fail to express the organ-specific marker ANT1. Thus, it appears that in bob1 mutants, the meristem domain is expanded at the expense of the cotyledon domains. The wild-type function of the BOB1 gene then is to promote organ fate and to limit meristem fate. BOB1 is expressed throughout the embryo during the early stages of embryogenesis when these developmental domains are being established. Because BOB1 mRNA expression is not itself restricted to any portion of the embryo, and assuming that BOB1 protein is not posttranslationally modified or transported differentially during embryogenesis, the activity of this gene must enable the correct establishment of patterning information without being part of the pattern itself.
Some pattern elements appear to develop normally in bob1 mutant embryos. The epidermis appears to be normally specified, as evidenced by expression of the ATML1 gene and exclusion of the CUC2 gene. This is in contrast with the rpk1 toad2 mutants, which fail to express ATML1 in the protodermal layer of embryos that lack cotyledons (Nodine and Tax, 2008 A second pattern element that appears to develop normally in bob1 mutant embryos is the separation of the embryo proper into distinct apical and basal halves. This is supported by the finding that the CUC2 and STM genes are strictly limited to the apical domain of the embryo in both wild-type and bob1 mutants. These two normal pattern elements, dividing the globular embryo into an apical and a basal hemisphere and the establishment of an epidermis, both require early, stereotyped cell divisions in the embryo. These early cell divisions appear normal by inspection with DIC optics as well as in many of the sections that were made of bob mutant embryos. Thus, the defects in pattern that we observe are not due to dramatic differences in early embryonic cell division patterns. In fact, changes in SCARECROW and DR5 expression are observed before changes in cell morphology become apparent.
In our screen for mutants that aberrantly express the STM reporter, we also recovered globular arrest mutants expressing the STM reporter throughout both apical and basal halves of the embryo. The identification of these mutants in our screen illustrates that it is possible to abolish this restriction with what are likely to be single gene mutations. It is therefore significant that bob1 does not abolish this aspect of polarity. This is despite the fact that bob1 mutant embryos fail to properly localize auxin, as determined by the expression of the DR5 promoter throughout the embryo. This is a somewhat surprising result given that auxin has been implicated in establishing apical-basal patterning in the early embryo (Friml et al., 2003
In addition to its role in development of apical/basal polarity in the embryo, auxin has been proposed to play a role in partitioning the apical domain of the embryo (for review, see Jenik and Barton, 2005 It is possible that BOB1 acts in the same pathway as the auxin transporters and their regulators and that all five of these differences are differences in severity of the mutant phenotype, with bob1 showing the most extreme mutant phenotype. It should be noted that in all of the mutant combinations affected in auxin transport, there are many gene family members that remain intact and can presumably supply activity, so it is reasonable to assume that a complete deficiency in auxin transport has not yet been observed. However, our data do not rule out that BOB1 may play a role in an as yet described independent pathway that is necessary for apical partitioning and is independent from its effect on auxin localization.
Yet another set of observations involving auxin parallel the observations of the bob1 mutant phenotype. Uniformly high levels of auxin have been observed when Brassica juncea and wheat (Triticum aestivum) embryos were exposed to exogenous auxin. Under these conditions, uniformly high levels of auxin are established throughout the developing embryos, and the embryos fail to develop cotyledons. Like bob1 mutants, the treated embryos never develop past a stage that resembles globular stage (Fischer and Neuhaus, 1996
As in bob1 mutants, mutants homozygous for the tpl mutation also fail to form cotyledons (Long et al., 2002 The presence of auxin throughout the bob1 embryo is difficult to reconcile with the inability of bob1 embryos to make cotyledons, as high levels of auxin are associated with cotyledon formation in the apical portion of the embryo in wild-type embryos. It is possible that normal cotyledon development requires the establishment of a differential of auxin concentration rather than depending upon the measurement of a threshold amount of auxin. Alternatively, concentrations of auxin, while generally increased throughout the embryo, may not reach sufficiently high levels in bob1 mutants to trigger cotyledon formation.
BOB1 Encodes a Protein Homologous to A. nidulans NudC
The founding member of this conserved gene family, NudC, was identified in a screen for nuclear distribution mutants in A. nidulans (Osmani et al., 1990
The molecular functions of NudC proteins are not well understood; however, NudC genes have been shown to be essential in several organisms. The original Aspergillus nudC mutant is a viable temperature-sensitive allele (Morris, 1976
NudC domains are predicted to have a similar three-dimensional structure as the
If BOB1 functions as a protein chaperone in Arabidopsis, then the phenotypes in bob1 mutants might be explained by a decrease or lack of activity of the substrates of BOB1 chaperone activity. These substrates could include proteins required for polar auxin transport or signaling during embryogenesis but could also include other proteins involved in pattern formation in the early Arabidopsis embryo. In animal systems, chaperones such as HSP90 play an important role in signaling by, among other substrates, steroid hormones (for review, see review Richter and Buchner, 2001
Plant Growth Conditions Unless otherwise noted, all plants were grown in soil (Scott's Metro-Mix 200) under constant light at 24°C.
Mutagenesis and Screen
Clearing of Tissue
Mapping and Isolation of the BOB1 Gene
Amplification of the genes in the BOB1 region for sequencing was done using ExTaq DNA polymerase from Panvera, and PCR products were cloned using pGEM-T Easy T-vectors from Promega. DNA sequencing was performed by University of Wisconsin Biotechnology Center Sequencing Services. Sequences were assembled and manipulated using DNAStar software. Sequence comparisons were done with ClustalX (Jeanmougin et al., 1998
Gene Expression
In situ hybridization was performed as described by Long et al. (1996) For in situ hybridization experiments on bob1 embryos, siliques were collected from bob1/+ plants, fixed, and sectioned. Because siliques were collected from heterozygous individuals, the bob embryos examined were side-by-side on the slide with their wild-type siblings, providing a positive control for hybridization of probes.
Imaging bob1-1 was genotyped using primers bob1-1_dCAPS_F (5'-TGGGACTAAAGCACGGACTGTTG-3') and bob1-1_dCAPS_R (5'-AGTAGCAGTCATCAGGCTTCACAGACC-3') followed by digestion with MspI. The resulting PCR products were 200 bp (wild-type) and 226 bp (bob1-1). bob1-2 was amplified in a reaction containing primers bob1-2_F (5'-GACTTTCTCGGAGAACAGAGC-3'), bob1-2_R (5'-CCTCCTGGAGATTCTGGATCC-3'), and LBb1 (5'-GCGTGGACCGCTTGCTGCAACT-3'). The resulting PCR products were 630 bp (wild type) and 450 bp (bob1-2).
DR5-GFP Imaging
Accession Numbers
Supplemental Data
This work was funded by grants from the National Science Foundation. Matthew Evans and Enrico Magnani provided helpful comments on the manuscript. Nicole Newell provided technical assistance with embryo preparations.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: M. Kathryn Barton (kbarton{at}stanford.edu).
[W] Online version contains Web-only data.
[OA] Open access articles can be viewed online without a subscription. www.plantcell.org/cgi/doi/10.1105/tpc.108.065284 Received January 5, 2009; Revision received June 24, 2009. accepted July 9, 2009.
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ABSTRACT
INTRODUCTION
2 testing reveals that the hypothesis of 3/16 segregation of bob1 embryos must be rejected (P < 0.001) but that a 1/4 hypothesis cannot be rejected (P = 0.5). Therefore, removing STM function from bob1 mutant embryos does not rescue the embryo arrest phenotype; the defect in bob1 mutants is not simply the overexpression of STM. 
-crystallin/HSP20 family of small heat shock proteins (Garcia-Ranea et al., 2002
