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First published online March 4, 2003; 10.1105/tpc.010678 American Society of Plant Biologists SPL8, an SBP-Box Gene That Affects Pollen Sac Development in Arabidopsis
a Department of Molecular Plant Genetics, Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany 1 To whom correspondence should be addressed. E-mail huijser{at}mpiz-koeln.mpg.de; fax 49-0-221-5062-113
SQUAMOSA PROMOTER BINDING PROTEIN–box genes (SBP-box genes) encode plant-specific proteins that share a highly conserved DNA binding domain, the SBP domain. Although likely to represent transcription factors, little is known about their role in development. In Arabidopsis, SBP-box genes constitute a structurally heterogeneous family of 16 members known as SPL genes. For one of these genes, SPL8, we isolated three independent transposon-tagged mutants, all of which exhibited a strong reduction in fertility. Microscopic analysis revealed that this reduced fertility is attributable primarily to abnormally developed microsporangia, which exhibit premeiotic abortion of the sporocytes. In addition to its role in microsporogenesis, the SPL8 knockout also seems to affect megasporogenesis, trichome formation on sepals, and stamen filament elongation. The SPL8 mutants described help to uncover the roles of SBP-box genes in plant development.
Transcription factors generally are believed to play key roles in the control of tissue-specific gene expression. Because this is a process central to the differentiation of multicellular organisms, determining the function of transcription factors can provide important insights into eukaryotic development. Analysis of the Arabidopsis genome reveals 29 classes of transcription factors, 16 of which appear to be unique to plants (Arabidopsis Genome Initiative, 2000  ). One of these is characterized by the presence of a DNA binding domain referred to as the SQUAMOSA PROMOTER BINDING PROTEIN (SBP) domain and encoded by the SBP-box, a feature characteristic of the Arabidop-sis SQUAMOSA PROMOTER BINDING PROTEIN–LIKE (SPL) gene family (Cardon et al., 1997, 1999). The 16 SPL genes found in the Arabidopsis genome form quite a heterogeneous family, but subfamilies can be recognized based on their genomic structures and the sequences of the deduced proteins (Cardon et al., 1999; Arabidopsis Genome Initiative, 2000; http://www.uni-frankfurt.de/fb15/botanik/mcb/AFGN/Huijser.htm).
The first SBP-domain proteins, isolated from snapdragon (Klein et al., 1996
Despite the efforts of many groups to isolate and characterize an ever-increasing number of Arabidopsis mutants, none of the corresponding genes isolated and published to date has been found to represent an SBP-box gene. The only described SBP-box gene with a known mutant phenotype remains the LIGULELESS1 (LG1) gene of maize, isolated by Moreno and co-workers (1997)
To obtain a better insight into the role of SBP domain transcription factors in plant development, we exploited available transposon-mutagenized populations of Arabidopsis to search for insertions in SBP-box genes. We identified and isolated three such transposon insertion alleles representing the SPL8 gene. The corresponding mutant plants all exhibited a strong reduction in fertility, primarily as a consequence of abnormal cell differentiation within the developing anthers. Only a few putative transcription factors that control this precisely ordered differentiation process (Scott et al., 1991 Here, we report the characterization of three loss-of-function mutant alleles representing the SBP-box gene SPL8, which, like NZZ/SPL, affects the early stages of microsporogenesis and megasporogenesis.
Identification and Isolation of Transposon-Mutagenized SPL8 Alleles Through systematic screening using a PCR-based reverse genetics approach (Baumann et al., 1998 ), we searched for mutant alleles of SPL genes in an Arabidopsis Columbia-0 (Col-0) population mutagenized with the heterologous and autonomous transposable element En (Spm) from maize (ZIGIA population) (Wisman et al., 1998; Steiner-Lange et al., 2001). However, in the case of SPL8, we fortuitously identified a first insertion allele within this population through a forward genetics screen for photosynthetic mutants (Varotto et al., 2000; Pesaresi et al., 2001). The plant line 5ATA20 exhibited a marked reduction in the effective quantum yield of photosystem II (P. Pesaresi and D. Leister, unpublished data). Furthermore, this line was found to harbor multiple copies of the transposon, and sequencing of their flanking genomic DNA revealed one of these to be inserted at the SPL8 locus, 1101 bp downstream of the presumed translation start codon and residing in the SBP domain coding sequence of the second exon (Figure 1). However, subsequent segregation analysis revealed that this insertion allele of SPL8 (referred to below as spl8-2; note that the alleles described in this article are numbered according to the order of their positions within the locus) could not be responsible for the observed reduced photosynthetic performance. Additional PCR-based screening of the ZIGIA population allowed the isolation of a second SPL8 mutant allele (referred to as spl8-3). In the corresponding line 7AT39, the insertion also was found in the SBP-box, but this time in an opposite orientation and 78 bp downstream of the site of insertion found in line 5ATA20 (Figure 1).
Finally, a third SPL8 insertion allele (spl8-1) was identified after screening the SINS database of transposon flanking sequences derived from the SLAT collection generated at the Sainsbury Laboratory (Norwich, UK) by Jones and co-workers (http://www.jic.bbsrc.ac.uk/sainsbury-lab/jonathan-jones/SINS-database/sins.htm). The SLAT collection represents pools of seeds obtained from a population of Arabidopsis plants mutagenized with a nonautonomous derivative, dSpm, of the maize transposable Spm/En element (Tissier et al., 1999 ). From the corresponding pool of seeds, we were able to identify two plants that both contained a transposon 167 bp downstream of the presumed start codon in the first exon but upstream of the SBP-box of SPL8 (Figure 1). DNA gel blot analysis, using right and left end border sequences of En, confirmed that one of these plants contained a unique transposon insertion in the SPL8 gene, whereas the other was found to harbor a second insertion in its genome. Selfing these plants resulted in progeny from which an spl8-1 homozygous mutant plant was selected to be the progenitor of the spl8-1 mutant lines 30213 and 30214. The homozygous spl8 mutant lines obtained from the ZIGIA population and the SLAT collection all showed the same deviations, in particular reduced fertility, as described below, from the wild-type phenotype. Segregation analysis of heterozygous plants showed a 3:1 wild type:mutant ratio, reflecting the recessive nature of the spl8 mutant alleles. Furthermore, F1 plants obtained from allelic tests among the different spl8 mutant lines again displayed these deviations, proving that the spl8 mutations are responsible for the observed phenotype. In all three mutant alleles, the En/dSpm insertions were expected to cause a premature translational stop of the predicted SPL8 gene product, as depicted in Figure 1. In fact, reverse transcriptase–mediated PCR performed on RNA extracted from inflorescences did not allow the detection of SPL8-derived transcripts in homozygous spl8-1 mutants. However, very low levels of transcripts of wild-type length were detected in both spl8-2 and spl8-3 plants (data not shown). This finding could be explained by assuming a low frequency of excision of the autonomous En element residing at the SPL8 locus in these two lines.
In the absence of an activator to encode a transposase, the dSpm element in line 30213 (i.e., representing the spl8-1 allele) is expected to remain stably integrated (Tissier et al., 1999 To avoid any influence of secondary site mutations on the analysis of the spl8 mutant phenotype, the spl8-1 mutant line was selected for further detailed histological analysis, as described below. However, we first characterized all three lines to confirm that they display the same major phenotypic aberrations.
Characterization of the spl8 Mutant Morphology
The 12 earliest flowers and siliques from the main inflorescences of an spl8 mutant plant and a wild-type plant, both at the same developmental stage and comparable to the plants shown in Figure 2A, are lined up in Figure 2B. Up to stage 13 of flower development (Smyth et al., 1990 ), spl8 flowers were almost indistinguishable from wild-type flowers, except that they appeared somewhat more slender. However, whereas wild-type siliques after stage 16 clearly elongated, the gynoecia of early spl8 mutant flowers did not change in length. A closer look with the scanning electron microscope revealed that in developing flowers, the spl8 anthers and their filaments remained smaller than those of the wild type (Figures 3A and 3B). As a result of their reduced size, the spl8 mutant stamens, four long and two short as in the wild type, did not overgrow the pistil, as is typical for wild-type flower development (i.e., at stage 14). At stage 13 to 14 of flower development, spl8 mutant anthers underwent dehiscence, but they always released fewer pollen grains than wild-type anthers (Figures 3C and 3D).
To quantify the degree of semisterility in spl8 mutants, we determined seed set and pollen production compared with those of wild-type plants (Table 1). The first five to seven flowers in the primary inflorescence of spl8 mutants grown under long-day conditions generally did not set seed (in secondary inflorescences, the number of flowers remaining seedless was even higher). In the wild type, occasionally only the first formed flower remained seedless. Flowers formed at later stages of inflorescence development in spl8 did set seed but fewer of them compared with the wild type (Table 2). It should be noted that in the wild type during the final stage of inflorescence development (i.e., before the complete cessation of flower formation), seed production seemed to decrease again, probably as a result of a diminishing supply of nutrients.
Seed set of spl8 flowers at defined positions in the primary inflorescence correlated with pollen production, with early flowers producing less pollen than later flowers (Table 1). This correlation also held true for the wild type, but significantly more pollen was produced compared with that in spl8 mutants. As a consequence of the strong reduction in pollen production, possibly together with the reduced length of the filaments, the frequency of self-fertilization of spl8 mutant flowers was reduced dramatically or did not occur at all, as was generally the case for the first flowers formed (Table 1). Manual pollination of homozygous spl8 mutant plants with their own pollen improved seed set. However, even when manually pollinated without emasculation, so as not to damage the flowers, seed set in the spl8 mutants remained low compared with that in the wild type (Table 2). That this is not simply the result of reduced numbers and/or viability of spl8 mutant pollen is suggested by the fact that cross-pollination with wild-type pollen also did not increase seed set to wild-type levels (Table 2). Furthermore, we determined the average number of seeds produced by emasculated wild-type flowers after cross-pollination with either mutant or wild-type pollen. This resulted in 46.5 (SD = 13.1, n = 6) seeds after pollination by the wild type and 31.3 (SD = 19.8, n = 6) and 43.5 (SD = 14.2, n = 6) seeds when pollinated by spl8-1 and spl8-2 mutants, respectively. These values do not differ significantly (t test; P > 0.1) from each other and strongly suggest that additional factors affect the overall fertility of spl8 mutant flowers (see Histological Analysis of spl8 Mutant Ovule Development below).
Histological Analysis of spl8 Mutant Anther Development Soon after stamen initiation, at stage 2 of wild-type anther development, archesporial cells arise from four small groups of hypodermal (L2 layer) cells at the corners of the anther primordium. Further mitotic divisions of these cells result in an anther with two theca, each of which bears a pair of pollen sacs or microsporangia. By the time the wild-type anther has reached stage 5 of development, each microsporangium has differentiated into a group of sporocytes surrounded by a tapetum, a middle cell layer, and an endothecium. At stage 6, wild-type sporocytes enter meiosis and form tetrads of microspores surrounded by a callose wall (stage 7), and the anther increases notably in size. Compared with that in the wild type, the initiation of microsporogenesis already was disturbed in the spl8-1 mutant. Archesporial cells failed to be formed at all four positions of the anther. As a consequence, spl8-1 mutant anthers sometimes developed with fewer than four differentiated pollen sacs (Figure 4A). Also, the subsequent histogenesis of the microsporangia that are formed in spl8 mutant anthers proceeded abnormally. The division of the outer secondary parietal cell layer remained incomplete, although sporadic, presumptive middle layer and endothecium cells were visible (Figure 4B).
The failure of sporogenous cells to undergo meiosis, observed after stage 5, also may contribute to the strong reduction of spl8 male fertility. However, without understanding the mechanism of SPL8 action on anther differentiation and development, it is difficult to determine whether the disruption in the microsporangium wall layers causes sporogenous cell abortion or sporogenous cell abortion leads to disruption in the microsporangium wall layers. Whereas in the subsequent stages (i.e., 7 and upward) the wild-type microspores were released from the tetrads and continued differentiation, the spl8 sporogenic tissue degenerated, as deduced from the large vacuoles that appeared in the tapetal cells and the dense staining of the sporogenous cells (Figure 4C). As a result, the "mature" spl8 mutant anthers appeared shorter and narrower than the wild-type anthers. However, occasionally, callose deposition was seen in some developing pollen sacs of the spl8 mutant after stage 5 of anther development (cf. with the wild type in Figure 4D). This finding indicates that some sporocytes initiate meiosis and continue their development (Figure 4E), which agrees with the observation that spl8 mutant anthers may release some pollen.
Histological Analysis of spl8 Mutant Ovule Development
As noted above, even after manual pollination, spl8 mutant flowers produced fewer seeds than wild-type flowers. A degeneracy of approximately half of the spl8 ovules may explain this discrepancy in seed set, assuming that degeneration of ovules in the wild type must be rare, because we did not observe this phenomenon in sections of wild-type flowers. However, the frequency with which abnormal ovule development was observed in spl8-1 mutant flowers seemed to vary, as deduced from the few spl8-1 mutant flower buds that were analyzed by sequential sectioning of the entire pistil. In the most extreme case, all of the ovules within the spl8-1 bud displayed a degenerate megaspore mother cell. In other samples, fewer than one-third of the ovules were affected. Like the production of pollen, this probably correlates with the position of the flower within the inflorescence. This aspect has not been investigated further.
Temporal and Spatial Expression Patterns of SPL8
We were able to identify and isolate three mutant spl8 alleles from two different transposon-tagged Arabidopsis populations. The phenotypes of the corresponding homozygous mutant plants all exhibited a strong reduction in male fertility. Together with the outcome of the heteroallelic tests and the segregation analyses after backcrossing to the wild type, these observations allowed the conclusion that the phenotype described is attributable to the mutation of the SPL8 gene and of SPL8 alone. This finding enabled us to determine a developmental role for a member of the Arabidopsis SBP-box gene family.
SPL8 Has a Major Effect on Sporogenesis
To what extent the developmental fate of sporogenous cells and parietal layers are interdependent remains unclear, but SPL8 has been found to be transcriptionally active in both tissues. Furthermore, the observation that tapetal ablation (Mariani et al., 1990
Another highly interesting phenotypic aspect of the spl8 mutant is the failure of initiation of all four microsporangia within the developing anther. Transverse sections through the early flower buds of spl8 mutants showed a complete absence of differentiated microsporangia in the greatly reduced anthers, whereas other transverse sections detected one or two partially developed microsporangia. This observation suggests that SPL8 somehow is involved in controlling the sensitivity of a yet unknown signal for pattern formation within the anther. In this context, it is interesting that SPL8 expression itself was found to be inducible by the phytohormone gibberellin (M. Schmid and D. Weigel, personal communication). Mutants known to be affected in either the biosynthesis or the perception of gibberellin often display severe fertility problems (Koornneef et al., 1985 spl8 mutants displayed a variable degree of expression, probably as a result of endogenous and environmental factors (e.g., the position of the flower in the inflorescence and photoperiod). However, and despite the fact that a complete loss of function will result in dramatically reduced progeny and thus a strong reduction in reproductive fitness within an Arabidopsis population, SPL8 function is not essential to Arabidopsis ontogenesis. All spl8 mutants remained capable of producing functional pollen and of setting viable seed. An obvious explanation would be a functional redundancy derived from another SBP-box gene. As mentioned above, SPL8 is one of 16 SBP-box genes found in the Arabidopsis genome. However, sequence alignment of their deduced protein products (data not shown) did not reveal a homolog (i.e., a possible SPL8 paralog). In fact, outside of its SBP domain, SPL8 does not display any obvious sequence similarity to other Arabidopsis SBP domain proteins. Thus, the possibility of another Arabidopsis SBP-box gene compensating for an SPL8 loss of function seems unlikely, but as long as the mode of action of SBP domain proteins remains unknown, this possibility cannot be excluded.
SPL8 Displays Only Limited Sequence Conservation Outside of the SBP Domain
Besides the DNA binding SBP domain, with its nuclear localization signal, a Basic Local Alignment Search Tool (BLAST) search of sequences available in the electronic databases did not indicate other known conserved domains that could be of help in understanding the mechanism of SPL8 function. However, almost immediately upstream of the SBP domain, SPL8 shares a short stretch of amino acid residues, RIGLNLGRTYF, with a few SBP domain proteins known from other species, such as AMSBPH3 from snapdragon and ZMSBP1, ZMSBP3, and ZMSBP4 from maize. Interestingly, part of this domain, GLNLGRTYF, also is conserved in the maize LG1 protein, the only other SBP domain protein with a known loss-of-function phenotype. LG1 controls the formation of the ligule, a structure typical of the leaves of grasses. In a search for the function of a possible LG1 ortholog in dicots, Mooney and Freeling (1997)
Plant Material and Growth Conditions Arabidopsis thaliana plants from ecotype Columbia-0 (Col-0) were grown in a phytochamber (CMP 3244; Conviron, Winnipeg, Canada) in plastic trays filled with ready-to-use commercial, prefertilized soil mixture (type ED73; Werkverband). For stratification, seeds were kept on wet filter paper for 4 days at 4°C in the dark before transferring to soil. Growing conditions were 22°C, 50% RH, and  150 µE·m-2·s-1 light (fluorescent Sylvania F72T12 cool-white light [75%] and incandescent Sylvania 100-W lamps [25%]). Cultivation was under a 16-h-light/8-h-dark (long-day) photoperiod regime.
Genomic DNA and RNA Isolation Total RNA for the detection of SPL8 transcripts was isolated from young inflorescences using the Qiagen RNeasy kit (Valencia, CA) according to the manufacturer's protocol Plant+Fungi. Before use for reverse transcriptase–mediated (RT) PCR, the RNA was treated with DNaseI (Roche, Mannheim, Germany) and purified subsequently over RNeasy mini spin columns (Qiagen). RT-PCR was performed using the Qiagen OneStep RT-PCR kit, according to the manufacturer's instructions, in combination with the following primer oligonucleotides: 5'-TCCATCGTTCTCTCCGGAGATGAAAG-3' (GC420; SPL8 forward primer) and 5'-GAATTTGAAGACGAAGACGCTGACGTG-3' (GC370; SPL8 reverse primer). Three-step cycling conditions were as follows: 26 PCR cycles at 94°C for 40 s, 65°C for 30 s, and 72°C for 1 min using 100 ng of RNA.
Mutant Isolation and Molecular Genetic Analysis The SLAT line 30213, which carries the spl8-1 allele, and the ZIGIA line 5AT20, which carries the spl8-2 allele, were back-crossed one time to the Col-0 wild type. The described mutant phenotype recurred only in F2 plants, segregating in a Mendelian fashion (i.e., 3:1 wild type:mutant). All of the mutants, but none of the wild-type-looking F2 plants, were found to be homozygous for the respective spl8 mutation. For the allelic test cross, homozygous spl8-1 and spl8-3 were crossed together. The resulting F1 generation displayed the semisterile phenotype of its parents. To discriminate both mutant alleles and to confirm the heteroallelic nature of the F1 plants, PCR analysis with the following oligonucleotide primer combinations was used: 5'-AGAAGCACGACGGCTGTAGAATAGGA-3' (En205) together with 5'-AACAACGACCGCCGTCACATCACC-3' (GC326) to identify the spl8-1 allele and En205 together with 5'-AAGACAGAAGTGGATTTCACGTCCAAC-3' (GC637) or 5'-GAGCGTCGGTCCCCACACTTCTATAC-3' (En8130) together with 5'-GAATTTGAAGACGAAGACGCTGACG-3' (GC370) to identify spl8-3. Oligonucleotides used for the PCR-based reverse genetics screen were 5'-TTGTTCTCCTACGACCAGACAGGACC-3' (GC633) and 5'-TTG-TCGAATTCCGACAGCAAATGGAAC-3' (GC634).
Oligonucleotide Synthesis, DNA Sequencing, and Sequence Analysis
Microscopy and in Situ Hybridization For scanning electron microscopy, freshly dissected flowers were mounted on aluminum specimen stubs using Tissue-Tek OCT compound (Sakura Finetek, Tokyo, Japan) and immediately shock frozen in liquid nitrogen. The samples were transferred subsequently to a Zeiss DSM 940 electron microscope (Jena, Germany) equipped with a cryo-chamber (Oxford Instruments). After sublimation of possible ice on their surfaces, the samples were sputter coated with gold and examined at an accelerating voltage of 5 kV.
For light microscopic examination of anthers, young flower buds ranging in length from 0.4 to 1.4 mm were fixed, dehydrated, and embedded as described by Sorensen et al. (2002)
Tissue preparation and in situ hybridization with digoxigenin-labeled RNA were performed according to the protocol published by Huijser et al. (1992) Both the in situ hybridization slides and the semithin sections were examined and photographed with a Zeiss Axiophot microscope equipped with differential interference contrast optics and a digital camera (Intas).
Remaining Techniques and Methods Upon request, all novel materials described in this article will be made available in a timely manner for noncommercial research purposes.
Accession Numbers
We are grateful to Susanne Höhmann and Sandra Kröber for excellent technical support and to all members of the laboratory for helpful discussions. We also thank Markus Schmid and Detlef Weigel for providing information concerning the gibberellin inducibility of SPL8 transcription. Part of this work was supported by the Deutsche Forschungsgemeinschaft. M.G. was funded by an Alexander von Humboldt fellowship.
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.010678. Received January 22, 2003; accepted February 6, 2003.
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Abstract
INTRODUCTION
