Plant Molecular Biology 53: 163–173, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands.
163
Conservation and divergence of ASK1 and ASK2 gene functions during male meiosis in Arabidopsis thaliana Dazhong Zhao1 , Tianfu Han1,3 , Eddy Risseeuw2 , William L. Crosby2,4 and Hong Ma1,∗ 1 Department of Biology and Huck Institute for Life Sciences, 315 Wartik Laboratory, Pennsylvania State University,
University Park, PA 16802, USA (∗ author for correspondence; e-mail
[email protected]); 2 Gene Expression Group, Plant Biotechnology Institute, National Research Council, Saskatoon SK S7N 0W9, Canada; present addresses: 3 Institute of Crop Breeding and Cultivation, The Chinese Academy of Agricultural Sciences, Beijing 100081, China; 4 Department of Computer Science, University of Saskatchewan, 1C101 Engineering, 57 Campus Drive, Saskatoon, SK S7N 5A7, Canada Received 24 June 2003; accepted in revised form 4 September 2003
Key words: Arabidopsis thaliana, ASK1, ASK2, male meiosis, SCF complex
Abstract Selective proteolysis of regulatory proteins mediated by the ubiquitin pathway is an important mechanism for controlling many biological events. The SCF (Skp1-Cullin-F-box protein) class of E3 ubiquitin ligases controls the ubiquitination of a wide variety of substrates, thereby mediating their degradation by the 26S proteasome. The Arabidopsis genome contains 21 genes encoding Skp1-like proteins that are named as ASKs (Arabidopsis Skp1-like). So far, only the ASK1 gene has been characterized genetically, and is known to be required for male meiosis, flower development, and auxin response. The ASK2 gene is most similar to ASK1 in terms of both the amino acid sequence and expression pattern. To compare ASK2 with ASK1 functionally in male meiosis, different transgenic lines over-expressing ASK1 and ASK2 were tested for their ability to complement the male meiosis defect of the ask1-1 mutant. The genomic ASK1 rescued the ask1-1 mutant defects. The 35S::ASK1 transgene restored male fertility to the ask1-1 mutant, although the percentages of normal pollen grains and tetrads were reduced. 35S::ASK2 lines in the ask1-1 background exhibited partial fertility with even fewer normal pollen grains and tetrads than those of the 35S::ASK1 lines. Detailed analysis of chromosome behavior during male meiosis demonstrated that 35S::ASK1 and 35S::ASK2 lines had different fractions of pollen mother cells undergoing normal meiosis. Our results suggest that ASK2 partially substitutes for ASK1 if expressed at higher than normal levels. Abbreviations: APT1, ADENINE PHOSPHORIBOSYLTRANSFERASE1; ASK, Arabidopsis Skp1-like; Cul1, cullin1; DAPI, 4 ,6-diamidino-2-phenylindole; SCF, Skp1-Cullin-F-box protein
Introduction Regulated proteolysis mediated by the ubiquitindependent pathway is a major mechanism for controlling many cellular events, such as cell cycle regulation, signal transduction, and transcription (Hershko and Ciechanover, 1998; Zheng et al., 2002). The ubiquitination process involves a three-enzyme cascade (E1, E2, and E3) which specifies substrate proteins for subsequent degradation by the 26S proteasome. Ubiquitin is first activated by the E1 enzyme, and then
transferred to the E2 ubiquitin-conjugating enzyme. In the third step the ubiquitin moiety is bound to a lysine residue of the substrate protein with the help of the E3 ubiquitin ligase, which confers substrate specificity. The SCF (for Skp1, Cullin/CDC53, F-box protein; also containing a fourth subunit called Rbx1) complexes form the largest known class of E3 ubiquitinprotein ligases (Schulman et al., 2000; Zheng et al., 2002). SCFs are involved in the ubiquitination of key proteins controlling fundamental biological processes, including cell cycle progress and transcriptional con-
164 trol (Hershko and Ciechanover, 1998; Koepp et al., 1999; Schulman et al., 2000; Gagne et al., 2002). Examination of the crystal structure of a human SCF complex containing the F-box protein Skp2 reveals that Skp1 acts as an adaptor that links cullin1 (Cul1) to the F-box domain of Skp2. Cul1 consists of two domains, a long stalk domain binding to Skp1 and a globular domain connecting to Rbx1 (Zheng et al., 2002). Rbx1 has a RING-H2 domain and interacts with the E2 enzyme (Kamura et al., 1999; Seol et al., 1999; Gray et al., 2002). The F-box domain is usually located at the N-terminus of F-box proteins and is required for binding to Skp1 (Skowyra et al., 1997; del Pozo and Estelle, 2000; Gagne et al., 2002; Risseeuw et al., 2003). The C-terminal portion of F-box proteins is responsible for recruiting substrates through protein-protein interaction. In Arabidopsis, the Skp1 homologue ASK1 (Arabidopsis Skp1-like1) has been found in SCF complexes involved in several regulatory processes. The Arabidopsis SCFTIR1 complex, consisting of ASK1, the cullin AtCUL1 and the F-box protein TIR1, regulates auxin response (Ruegger et al., 1998; Gray et al., 1999). Recent studies demonstrate that the degradation of the AUX/IAA family of repressor proteins is dependent on SCFTIR1 (Gray et al., 2001) and on the COP9 signalosome (Schwechheimer et al., 2001). COI1 is another F-box protein that mediates jasmonate response, and it physically associates with AtCUL1, AtRbx1, and either ASK1 or ASK2 (Xie et al., 1998; Xu et al., 2002). In addition, the F-box protein UFO controls flower development and interacts with ASK1 genetically and physically to regulate expression of the floral homeotic genes AP3 and PI (Ingram et al., 1995; Levin and Meyerowitz, 1995; Samach et al., 1999; Zhao et al., 1999, 2001). In yeast and man, there is only one known functional SKP1 gene. The single yeast SKP1 gene is known to be essential for mitosis and regulates transcription factors. However, the Arabidopsis genome possesses 21 known or predicted ASK genes (AGI, 2000; Farras et al., 2001; Risseeuw et al., 2003; Zhao et al., 2003). Similarly, other plants, as well as Drosophila and Caenorhabditis elegans, also have at least several Skp1 homologues. The presence of multiple Skp1 homologues makes it possible for two or more genes to act redundantly while, at the same time, divergence of gene functions may occur in other homologues through gene evolution. Functional comparison allows us to determine which ASK genes share the same functions and which have diverged.
Among the 21 ASK genes in the Arabidopsis genome, only ASK1 has been characterized genetically. In contrast to yeast skp1 mutants, which are lethal, the ask1-1 null mutant is viable during both vegetative and reproductive development. At the amino acid sequence level, ASK1 is most similar to ASK2 with 75.4% identity and 84.8% similarity in amino acid sequence. Like ASK1, ASK2 has been shown to associate with F-box proteins (Gray et al., 1999; Samach et al., 1999; Risseeuw et al., 2003). Furthermore, ASK1 and ASK2 genes have nearly identical expression patterns, although ASK2 has lower expression levels than ASK1 (Porat et al., 1998; Zhao et al., 2003; Risseeuw and Zhao, unpublished data). Therefore, ASK1 and ASK2 may have partially redundant functions. In addition to its involvement in hormone response and flower development, ASK1 is also required for male meiosis and fertility (Yang et al., 1999). The ask1-1 mutant is defective in homologue separation, resulting in the failure of some homologues to separate at anaphase I. The ask1-1 mutant produces polyads containing microspores of variable number and size, leading to non-viable pollen grains and male sterility. This severe ask1-1 phenotype in male fertility, in contrast to the other mild aspects of the ask1-1 phenotype, suggests that ASK2 is unable to provide the needed function during male meiosis. It is possible that the level of ASK2 expression is too low or that the ASK2 protein has a different function. To investigate these non-mutually exclusive possibilities, we compared transgenic Arabidopsis plants that over-expressed ASK1 or ASK2 in an ask1-1 mutant background. Our analyses of fertility, pollen development, and male meiosis among different transgenic lines demonstrate that elevated ASK2 expression can partially suppress the ask1-1 defect in male meiosis. This indicates that the ASK2 protein is sufficiently different from ASK1 and is unable to fully replace ASK1 function during meiosis. Therefore, ASK1 and ASK2 exhibit functional conservation and divergence during male meiosis in Arabidopsis. Materials and methods Plant materials and growth conditions The wild-type, ask1-1 mutant and transgenic lines are of Landsberg erecta or Wassileskija ecotypes. Plants were grown in Metro-Mix 200 soil (Scotts-Sierra Horticultural Products Co., Maryville, OH) at 22 ◦ C with
165 Selection of transgenic lines
Figure 1. A diagram of constructs and expression analysis of the wild-type, ask1-1 mutant and transgenic lines. A. A diagram of genASK1, 35S::ASK1 and 35S::ASK2 constructs. B. RT-PCR results of ASK1 expression in the wild-type (lane 1), ask1-1 mutant (lane 2), genASK1#1 (lane 3) and 35S::ASK1#1 (lane 4) transgenic lines. C. RT-PCR results of ASK2 expression in the wild-type (lane 1), ask1-1 mutant (lane 2) and 35S::ASK2#2 lines (lane 3). The APT1 gene was used as a control.
16 h of light and 8 h of darkness. For the mutant background selection, Murashige and Skoog (MS) plate containing 50 µg/ml kanamycin was used to grow seedlings. Plasmid construction and plant transformation The constructs of genomic ASK1 driven by its own promoter (genASK1), and the ASK1 and ASK2 cDNAs driven by the cauliflower mosaic virus 35S promoter (35S::ASK1 and 35S::ASK2) are illustrated in Figure 1. Details of the construction of these plasmids will be described elsewhere (Risseeuw, unpublished data). In addition to the ASK genes, the constructs contain the selectable phosphinothricin N-acetyltransferase gene conferring resistance to the herbicide phosphinothricin (Liberty Herbicide; AgroEvo USA Company). Plant transformation plasmids were introduced into Agrobacterium tumefaciens TCL 200 by heat shock. Agrobacterium-mediated transformation of Arabidopsis was performed essentially according as described previously (Clough and Bent, 1998).
T0 seeds were collected, dried and then sown in the soil. The seedlings at the cotyledon stage were sprayed with Liberty Herbicide (500 µl/l) and selected for resistant plants. Pollen from the herbicideresistant plants was used to pollinate the ask1-1 mutant. F1 and F2 plants were selected again for herbicide resistance. F3 seeds were collected from 20 herbicide-resistant F2 plants with normal or partial fertility. Homozygosity of the ask1-1 allele in the F2 plants was determined by segregation analysis of F3 plants for the presence of the Ds insertion carrying the KanR marker on MS medium containing kanamycin. The ask1-1 genotype was further confirmed by PCR with primers of oMC 221 (5 AAGGTGATCGAGTATTGCAAGAG-3), oMC 383 (5 -GAAGATAGTCATGATTCATGAAG-3) and Ds5-2 (5 -CGTTCCGTTTTCGTTTTTTACC-3) (Zhao et al., 1999). RT-PCR Total RNA was extracted from young inflorescences of F2 plants using a Qiagen RNeasy Plant Mini Kit (Valencia, CA) according to the manufacturer’s instructions. Total RNA (1 µg) was treated with DNase I in 10 µl of reaction and then was reversedtranscribed with an Oligo-dT primer and the Superscript RT II Kit (Life Technologies/Gibco-BRL, Carlsbad, CA). The reaction was diluted 10 times and 4 µl was used as template for 25 cycles of PCR in a 20 µl reaction. The gene-specific primers for the ASK1 gene were oMC 221 and oMC 383; the primers for the ASK2 gene were oMC 420 (5 GGATCCGAAACCACGGCCGAT-3 ) and oMC 593 (5 -AAATGGGTCGAGGACATGAC-3). The APT1 (ADENINE PHOSPHO-RIBOSYLTRANSFERASE1) gene was used as a control and the primers for this gene were oMC 571 (5 -TCCCAGAATCGCTAAGATT3 ) and oMC 572 (5 -CCTTTCCCTTAAGCTCTG-3 ) (Moffatt et al., 1994). PCR products were separated on a 1.2% agarose gel. Phenotypic characterization F2 plant flowers were examined under a Nikon dissecting microscope (Nikon Stereoscopic Microscope, Model SMZ-U, Tokyo, Japan). Pollen, microspores, tetrads and chromosomes were observed under a compound microscope (Nikon Eclipse E800, Tokyo, Japan). All light microscopic images were photographed
166
Figure 2. Phenotypic analysis of the wild-type, ask1-1 mutant and transgenic lines. A. A wild-type flower showing pollen grains released from anthers. B. All wild-type pollen grains were viable, as indicated by red staining. C. Wild-type microspores were nearly uniform in size. D. Typical wild-type tetrads containing four equal-sized microspores. E. An ask1-1 mutant flower lacking released pollen. F. The ask1-1 mutant pollen grains were stained in blue, indicating that they were not viable. G. Microspores from the ask1-1 mutant with variable sizes. H. Two tetrads (arrows) from the ask1-1 mutant with irregular number and size of microspores. I–L. The genASK1#1 transgenic line showing normal pollen in the anther (I), pollen stained in red (J), microspores (K) and tetrads with four spores (L). M. A flower from the 35S::ASK1#1 transgenic line showing many pollen grains. N. Most pollen grains were stained in red in 35S::ASK1#1. O. Two microspores (arrows) having irregular sizes in 35S::ASK1#1. P. Four equal-sized microspores were observed in most tetrads in 35S::ASK1#1. Q. In the 35S::ASK2#2 transgenic line, some pollen grains were released from anthers. R. Some pollen grains were stained in red in 35S::ASK2#2. S. Variable-size microspores from the 35S::ASK2#2 line (arrows point to small spores). T. One tetrad (arrow) having abnormal number of microspores. A, E, I, M Q (bar 250 µm); B, F, J, N, R; C, G, K, O, S and D, H, L, P, T (bar 10 µm) have the same magnification.
167 with a digital camera. For pollen staining, inflorescences were fixed and stained as previously described (Alexander, 1969). The viable and dead pollen grains were numbered separately. To observe microspores and tetrads, fresh young anthers were dissected using a fine needle to release the contents onto a slide, then samples were stained with 0.02% Toluidine Blue. The analysis of male meiosis was performed as described previously (Ross et al., 1996), with the following modification. Digested anthers were covered by a cover glass and crushed by tapping gently. The slides were frozen on dry ice before removing the cover glass. Slides were dried by baking on a hot plate at 60 ◦ C for several minutes. DAPI (4 ,6-diamidino-2phenylindole; Vector Laboratories, Burlingame, CA) was applied and the slide was mounted by nail polish.
Results Generation of transgenic ASK lines To compare the function of ASK1 and ASK2 genes in male meiosis, we made three constructs for Agrobacterium tumefaciens transformation: genomic ASK1 driven by its own promoter (genASK1), and the ASK1 and ASK2 cDNAs driven by the cauliflower mosaic virus 35S promoter (35S::ASK1 and 35S::ASK2; Figure 1A). About 100 transformants were obtained for each construct by selection on soil for resistance to the herbicide phosphinothricin. None of the T1 transformants had morphological or fertility defects. Forty transgenic lines for each construct were crossed with the ask1-1 mutant to introduce the transgene into the mutant background. The ask1-1 mutation was caused by a Ds insertion that confers resistance to kanamycin; F2 progeny plants carrying both the ask1 mutation and one of the transgenes were selected by resistance to both kanamycin and phosphinothricin. Some of these were recognizable as being ask1 homozygotes for their reduced fertility and other phenotypes. In all cases, the ask1 homozygosity was verified by analyzing segregation of kanamycin resistance in F3 progeny and further confirmed by PCR (see Materials and methods). For phenotypic studies described hereon in this study, all transgenic plants with any of the three transgenes have the ask1-1 mutant background and will be referred to without explicitly stating the ask1-1 genotype. First, we examined the transgenic lines for male fertility as an indication of functional rescue of the
ask1 mutation. Sixteen of the 40 genASK1 lines were found to exhibit complete rescue of the ask1-1 mutant defects. Ten lines with the 35S::ASK1 transgene displayed complete or partial fertility. However, none of the transgenic lines with 35S::ASK2 had normal fertility, although they produced a small number of seeds. For each construct, the two lines with the highest fertility were selected for detailed analysis and will be referred to as genASK1#1, genASK1#2, 35S::ASK1#1, 35S::ASK1#2, 35S::ASK2#1, and 35S::ASK2#2. In the genASK1#1 line, the ASK1 transgene expression level in the inflorescence was comparable to that of the wild-type ASK1 allele, whereas the 35S::ASK1#1 line showed higher than normal ASK1 expression (Figure 1B). Furthermore, a greater than normal level of ASK2 expression was detected in the 35S::ASK2#2 line (Figure 1C). A control gene, APT1, has an expected constitutive expression (Moffatt et al., 1994). Therefore, 35S::ASK1 restores normal fertility to the ask1-1 mutant, but 35S::ASK2 cannot fully substitute for the absence of ASK1 function as assayed by fertility. Male fertility analyses of the wild-type, ask1-1 mutant and transgenic lines To further characterize the function of the ASK transgenes, pollen development was analyzed in more detail in the selected transgenic lines. Unlike the wild type (Figures 2A–D, 3 and 4), there was no pollen released from the anthers in the ask1-1 mutant (Figure 2E). Wild-type pollen grains are nearly uniform in size and stained in red (Figure 2B), which indicates viability. In contrast, pollen grains inside the ask1-1 anther were variable in size and not viable, as indicated by their blue color (Figures 2F and 3) (Yang et al., 1999). Different from the wild type (Figure 2C), the irregular size of the ask1-1 mutant microspores was apparent (Figure 2G). Moreover, the microspore numbers in the polyads were often abnormal (Figures 2H and 4), instead of the four microspores in normal tetrads (Figures 2D and 4). In the genASK1#1 line, the flowers were phenotypically normal with abundant pollen grains released from the anther (Figure 2I). Pollen staining showed that the pollen grains were viable (Figures 2J and 3). The genASK1#1 line also produced normal microspores and tetrads similar to the wild type (Figures 2K, 2L and 4). In the 35S::ASK1#1 line, the transgene was sufficient for normal flower development and abundant pollen production for male fertility (Figure 2M). Fur-
168
Figure 3. Comparison of viable pollen grains among the wild-type (1), ask1-1 mutant (2), genASK1#1 (3), 35S::ASK1#1 (4) and 35S::ASK2#2 (5) transgenic lines.
35S::ASK2#2 line produced four microspores (Figures 2T and 4), many spores from these meioses would be non-viable due to abnormal chromosome numbers resulting from defective chromosome segregation (see below). Taken together, our results indicate that the 35S::ASK2 transgene could partially rescue the pollen development defects in the ask1-1 mutant, suggesting that the ASK2 gene can substitute for ASK1 in meiosis to some extent. At the same time, the fact that 35S::ASK2 did not result in as high fertility as 35S::ASK1 indicates that the ASK2 gene cannot fully replace the ASK1 function. Male meiosis in the ask1-1 mutant and transgenic lines
Figure 4. Comparison of tetrads with four microspores of nearly equal sizes among the wild-type (1), ask1-1 mutant (2), genASK1#1 (3), 35S::ASK1#1 (4) and 35S::ASK2#2 (5) transgenic lines.
ther analysis indicates that most (55±26%) of the pollen grains produced from 35S::ASK1#1 were normal (Figures 2N and 3). However, some microspores were not uniform in size and the proportion of tetrads with four microspores (73±15%) was lower than that observed in the wild type (95±2%, Figures 2O, 2P and 4). Even when four microspores are formed in a tetrad, not all spores are normal because of chromosome segregation defects (see below). This provides an explanation why the percentage of meioses that produced four spores was greater than the percentage of viable pollen grains. In the 35S::ASK2 transgenic lines, the fertility defect of the ask1-1 mutant was only partially rescued, as indicated by the short seedpods compared to the 35S::ASK1 and genASK1 lines as well as the wild type (data not shown). In the 35S::ASK2#2 line, some of pollen grains were released from the anther, although fewer than those in the 35S::ASK1#1 line (Figure 2Q). Pollen staining indicated that 23±8% of the pollen grains were viable, lower than that in the 35S::ASK1#1 line (Figures 2R and 3, t = 2.39 > t0.05 = 2.11). Correspondingly, most of microspores were of variable size (Figure 2S). Although male meioses in the
To more directly examine the effect of the ASK transgenes on male meiosis, we investigated meiosis by means of chromosome spreading and DAPI staining techniques (Ross et al., 1996). The genASK1#1 line showed normal male meiosis similar to the wild type (Ross et al., 1996; Figure 5 and data not shown). During prophase I in the genASK1#1 line, at leptotene stage the chromosomes were observed as thin threadlike structures (Figure 5A), then at zygotene the homologues started to pair (Figure 5B), and at pachytene chromosomes condensed into fully synapsed homologues (Figure 5C). The desynapsing chromosomes at diplotene become even shorter and thicker (Figure 5D). At diakinesis, five highly condensed bivalents are formed with X-shaped structures (Figure 5E). The five bivalents align along the equator at metaphase I (Figure 5F). At anaphase I in genASK1#1, the homologues separate and five homologues migrate toward each opposite pole (Figure 5G), leading to the formation of two groups of five partially decondensed chromosomes at telophase I (Figure 5H). Although the ask1-1 mutant was shown to be defective in homologue separation by means of DAPI staining of whole cells (Yang et al., 1999), the analysis of its meiotic phenotype by chromosome spread has not been described. Our results are in general agreement with previous finding that the ask1-1 mutant does not have obvious defects before pachytene (data not shown). At metaphase I, five highly condensed bivalents were rarely observed, suggesting that chromosome condensation prior to metaphase I is abnormal (Figure 5K). In addition, during this stage, some condensed bivalents failed to align along the equator. Furthermore, homologue separation at anaphase I was
169
Figure 5. Comparison of male meioses among the ask1-1 mutant and transgenic lines. Shown are DAPI staining images of chromosomes. F, K, P, U: metaphase I (MI); G, L, Q, V, D : anaphase I (AI); H, M, R, W: telophase I (TI); I, N, S, X: metaphase II (MII); J, O, T, Y: anaphase II (AII); Z, A –C : telophase II (TII). A–J, Z. Meiosis in the genASK1#1 line is similar to the wild type; in prophase I at leptotene (A, thread-like chromosomes), late zygotene (B, more condensed chromosomes), pachytene (C, thicker chromosomes), diplotene (D, shorter and thicker chromosomes) and diakinesis (E, five X-shaped homologues), respectively. F. At metaphase I, five fully condensed homologues congress along the equator. G. At anaphase I, each group of five chromosomes moving towards opposite poles. H. Two groups of chromosomes at each pole at telophase I. I. At metaphase II showing that each five chromosomes distribute at each side of organelle band. J, Z. Four groups of five newly separated chromosomes were observed at anaphase II (J) and telophase II (Z). K–O, A . The ask1-1 mutant. K. Five bivalents at metaphase I. L. Anaphase I showing non-separated bivalents (arrows). M. Abnormal distribution of homologues at telophase I (arrows). N. Chromosomes at metaphase II exhibiting uneven distribution. O. Anaphase II chromosomes were not well separated nor condensed. A . The uneven distribution of chromosomes at telophase II. P–T, B . The 35S::ASK1#1 line, showing normal meiosis at metaphase I (P), anaphase I (Q), telophase I (R), metaphase II (S), anaphase II (T), and telophase II (B ). U–Y, C , D . The 35S::ASK2#2 line, exhibiting normal meiosis at metaphase I (U), anaphase I (V), telophase I (W), metaphase II (X), anaphase II (Y), and telophase II (C ); abnormal chromosome behavior at anaphase I (D ). All panels have the same magnification (bar 10 µm).
170 defective in the ask1-1 mutant (Figure 5L), similar to previous results (Yang et al., 1999). The defects in separation of homologues most likely result in the uneven distribution of chromosomes at telophase I (Figure 5M). At metaphase II, in the genASK1#1 line, ten condensed chromosomes are clearly visible and distributed equally to the two sides of organelle band into two groups of five each (Figure 5I). At anaphase II, the individual chromatids separate and move toward the spindle poles (Figure 5J). Finally, at telophase II, four groups of five chromatids are formed (Figure 5Z). In contrast, the ask1-1 homologues are distributed unevenly and the separation of chromatids is abnormal during meiosis II (Figure 5N, O), which results in the formation of abnormal numbers of chromosomal clusters, each of which contained a different number of chromosomes or chromosome fragments (Figure 5A ). In addition, at meiosis II, most of homologues and chromatids have sharp edges and an elongated shape, suggesting that chromosome condensation is defective (Figure 5N, O). Examination of male meiosis in the 35S::ASK1#1 and 35S::ASK2#2 lines showed that normal male meiosis occur at some frequency in both lines. In the 35S::ASK1#1 line, most of the male meiocytes had normal chromosome behavior during meiosis I (e. g. Figure 5P–R) and meiosis II (Figure 5S, T, B ). Normal meiosis also occurred in the 35S::ASK2#2 line (e.g. Figure 5U–Y, C ), but the number of cells with normal chromosome behavior in the 35S::ASK2#2 line was lower than that in 35S::ASK1#1. Some of the meiotic cells in 35S::ASK2#2 line (e.g. Figure 5D ) had chromosomal phenotypes similar to, but less severe than, those of the ask1-1 meiocytes. In summary, the ask1-1 mutant is defective in homologue segregation, which is consistent with our previous findings (Yang et al., 1999). The genASK1 transgene can restore meiosis defects to the ask1-1 mutant. Both of 35S::ASK1 and 35S::ASK2 transgenes can rescue male meiosis defects of the ask1-1 mutant, but the 35S::ASK2 transgene is less effective, suggesting that ASK1 and ASK2 do not play an equal role during male meiosis.
Discussion Among the ASK proteins, ASK2 is most similar to ASK1 in amino acid sequence, with an overall 75% amino acid sequence identity. The ASK2 gene also
has a similar expression pattern to that of ASK1 (Porat et al., 1998; Zhao et al., 2003; Risseeuw and Zhao, unpublished data). Furthermore, both ASK1 and ASK2 can interact with the same F-box proteins, such as TIR1, COI1 and UFO, in the yeast two-hybrid system, suggesting that ASK1 and ASK2 may play related roles in plant development and other processes (Gray et al., 1999; Samach et al., 1999; Xu et al., 2002; Risseeuw et al., 2003). In contrast, the fact that the ask1-1 mutant has multiple defects indicates that ASK1 and ASK2 functions are not completely redundant. ASK1 is expressed at a higher level than ASK2, particularly in the male meiocytes (Porat et al., 1998; Zhao et al., 2003). The increased levels of ASK2 expression from the 35S promoter could partially rescue the male fertility, pollen development and male meiosis defects in the ask1-1 mutant. Furthermore, 35S::ASK2 could largely restore normal rosette leaf size, plant stature, and floral structure to the ask1-1 mutant (Risseeuw and Zhao, unpublished data), suggesting that the inability of the endogenous ASK2 to substitute for ASK1 is in part due to its lower expression than ASK1. Therefore, the ASK1 and ASK2 proteins share similar functions in the control of plant development. On the other hand, the 35S::ASK2 transgene was not as effective as the 35S::ASK1 transgene in restoring pollen development and male meiosis to the ask1-1 mutant. Similarly, compared to ASK1, over-expression of ASK2 is less efficient in rescuing some of flower defects in the ask1-1 mutant (Risseeuw and Zhao, unpublished data). These observations suggest that the ASK2 protein is not identical to ASK1 in function during plant development, particularly in controlling male meiosis. In other words, the functional divergence of endogenous ASK1 and ASK2 genes are likely due to differences in both expression pattern and protein sequence. In known SCF ubiquitin ligases, Skp1 homologues connect the catalytic cullin/Rbx1 with F-box proteins, which bind substrates. ASK1 and ASK2 have both been found to be components of SCF ubiquitin ligases with the F-box proteins TIR1 and COI1. In addition, yeast two-hybrid experiments showed that ASK1 and ASK2 can interact with F-box proteins UFO and ORE9. The Arabidopsis genome is estimated to have more than 700 F-box proteins (Gagne et al., 2002), suggesting that ASK1 and ASK2 may interact with other unidentified F-box proteins. Though not known, it is reasonable to hypothesize that ASK1 interacts with one or more F-box proteins during male meiosis
171 to mediate the degradation of specific substrates. However, these studies did not provide any information on whether ASK1 and ASK2 bind to the same F-box proteins with the same or different affinities. Skp1 and its homologues share two highly conserved regions with a variable linker in between. X-ray crystallographic analyses of the dimer of the human Skp1 protein with the F-box protein Skp2 and the SCF complex of human SKP1, SKP2, Cullin1, and Rbx1 have yielded three-dimensional structures of these complexes (Schulman et al., 2000; Zheng et al., 2002). These structures revealed that the N-terminal conserved region of Skp1 provides much of the interaction with Cul1 and the C-terminal region of Skp1 contacts the F-box of the F-box protein. The N and C termini of ASK1 and ASK2 are very similar and have highly conserved residues corresponding to the functionally important amino acids in human Skp1. This is consistent with the observation that ASK1 and ASK2 can both interact with several F-box proteins. Nevertheless, it is possible that minor differences in ASK1 and ASK2 sequences might have led to slight difference in binding affinity to F-box proteins. Alternatively, ASK1 and ASK2 may bind to cullin and F-box proteins to form SCF complexes that have slightly different distance between the substrate and the catalytic portion of the SCF complex. It was proposed, based on the crystal structure, that the SCF complex may position the substrate for ubiquitination by the E2 ubiquitin-conjugating enzyme. Relevant to the idea of a difference in distance between the substrate and the catalytic site is the presence of an additional 10 residues in ASK2 between helix 3 at the end of the N-terminal and helix 4 of the C-terminal conserved regions. In the Skp1 protein, this divergent region corresponds to a structurally undefined ‘loop’ in between the helices 3 and 4 that interact with Cul1. This suggests that an insertion in this region might shift certain structural components of ASK2, relative to ASK1, thereby causing a change in the interaction with cullin and in the catalytic properties of SCF. It is possible that the putative substrate(s) of the ASK1SCF is (are) more sensitive to the spatial differences between ASK1 and ASK2 than the other substrates in vegetative and floral tissues. The ASK1 gene is widely expressed in the plant at very high levels (Porat et al., 1998, Zhao et al., 2003; Risseeuw and Zhao, unpublished data), suggesting that the ASK1 promoter is very strong, similar to the 35S promoter. This similarity is supported by the fact that the 35S::ASK1 transgene can fully restore
the rosette leaf size, plant stature, petal and stamen number to the ask1-1 mutant. However, in 35S::ASK1 transgenic lines, male meiosis was not completely normal, suggesting that the 35S promoter is not as strong as the ASK1 promoter in meiotic cells. This could explain the observation that in the inflorescence of 35S::ASK1 transgenic line the ASK1 gene expression level is higher than of wild type, but the male meiosis defect is not fully rescued. The idea that the meiotic defects in the 35S-ASK1 lines are due to insufficient ASK1 function is further supported by the observation that lines with some defects in vegetative and flower development also have more severe meiotic defects. Another formal possibility for the mild meiotic defects in the 35S::ASK1 transgenic lines is that the defects might be due to abnormally high levels of transgene expression in male meiocytes. However, this is not likely because the 35S::ASK1 transgene did not cause any defects in the ASK1 wild-type background. Although the 35S::ASK1 transgene could not fully restore the male meiosis to the ask1-1 mutant, sufficient ASK1 function was achieved to rescue enough pollen development necessary for male fertility, as indicated by the observation that some of 35S::ASK1 transgenic lines showed normal seed set. The rosette leaf size and plant stature are normal in the 35S::ASK2 transgenic lines. In addition, 35S::ASK2 lines are able to produce some functional and viable pollen. However, none of the 35S::ASK2 transgenic lines has normal fertility compared to the 35S::ASK1 transgenic lines. Furthermore, the pollen development and male meiosis are different between those two transgenic lines. Therefore, the 35S promoter was able to drive substantial meiotic expression and provide a system to compare the function of ASK1 and ASK2. A more definitive test for the relative levels of ASK1 and ASK2 would be to determine the levels of these proteins, for example by means of immunodetection. However, available antibodies against ASK1 or ASK2 all react with both ASK2 and ASK1, making quantification of each protein difficult. Nevertheless, previous studies of ASK1 and ASK2 proteins are consistent with their respective mRNA levels; therefore, there is no evidence of marked differences in translational efficiency or protein stability between these two proteins. Our analysis of ASK1 and ASK2 function in male meiosis suggests that these two genes are partially redundant and partially distinct. The Arabidopsis ASK gene family forms several subgroups of highly similar genes, including some that form tandem repeats,
172 suggesting that they are, in part, the result of recent duplications. Gene duplication followed by functional diversification is a well-established mechanism for gene evolution (Clegg et al., 1997; Knight, 2002). Immediately after gene duplication, the duplicated genes are likely to be functionally redundant. If they remain completely identical in function, then there is a high probability for gene silencing due to mutation. However, if gene function diverges, then selection can maintain both of the duplicated genes. ASK1 and ASK2 might be an example of such duplicated genes. Whereas much of the functions during vegetative development may be redundant between these two genes, the function of ASK1 in meiosis might be quite distinct. Whether ASK2 also has a function in meiosis distinct from ASK1 cannot yet be determined and awaits further genetic studies. It is possible that other groups of ASK genes might have similar relationships that are also worthy of investigation.
Acknowledgements We thank AgroEvo USA Company for providing Liberty Herbicide, and A. Omeis and J. Wang for plant care. We also thank E. Harris, M. Henry and N. Rigel for help with the identification of transgenic lines. We are grateful for comments on the manuscript from W. Hu, H. Kong, W. Li, W. Ni, L. Timofejeva, G. Wang, L. Zahn, and W. Zhang. This work was supported by grants from the US National Science Foundation (MCB-9896340; MCB-0092075) and from the National Institutes of Health (RO1 GM63871) to H.M., by the NRC-PBI core funding to E.R. and W.L.C., by a grant from US National Science Foundation 2010 program to W.L.C. (Grant 0115870), and by Funds from the Department of Biology and the Huck Institute for Life Sciences at the Pennsylvania State University. T.H. was supported by the China Scholarship Council and the National Natural Science Foundation of China.
References Arabidopsis Genome Initiative. 2000. Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408: 796–815. Alexander, M.P. 1969. Differential staining of aborted and nonaborted pollen. Stain Technol. 44: 117–122. Clegg, M.T., Cummings, M.P. and Durbin, M.L. 1997. The evolution of plant nuclear genes. Proc. Natl. Acad. Sci. USA 94: 7791–7798.
Clough, S.J. and Bent, A.F. 1998. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16: 735–743. del Pozo, J.C. and Estelle, M. 2000. F-box proteins and protein degradation: an emerging theme in cellular regulation. Plant Mol. Biol. 44: 123–128. Farras, R., Ferrando, A., Jasik, J., Kleinow, T., Okresz, L., Tiburcio, A., Salchert, K., del Pozo, C., Schell, J. and Koncz, C. 2001. SKP1-SnRK protein kinase interactions mediate proteasomal binding of a plant SCF ubiquitin ligase. EMBO J. 20: 2742–2756. Gagne, J.M., Downes, B.P., Shiu, S.H., Durski, A.M. and Vierstra, R.D. 2002. The F-box subunit of the SCF E3 complex is encoded by a diverse superfamily of genes in Arabidopsis. Proc. Natl. Acad. Sci. USA 99: 11519–11524. Gray, W.M., del Pozo, J.C., Walker, L., Hobbie, L., Risseeuw, E., Banks, T., Crosby, W.L., Yang, M., Ma, H. and Estelle, M. 1999. Identification of an SCF ubiquitin-ligase complex required for auxin response in Arabidopsis thaliana. Genes Dev. 13: 1678– 1691. Gray, W.M., Kepinski, S., Rouse, D., Leyser, O. and Estelle, M. 2001. Auxin regulates SCFTIR1 -dependent degradation of AUX/IAA proteins. Nature 414: 271–276. Gray, W.M., Hellmann, H., Dharmasiri, S. and Estelle, M. 2002. Role of the Arabidopsis RING-H2 protein RBX1 in RUB modification and SCF function. Plant Cell 14: 2137–2144. Hershko, A. and Ciechanover, A. 1998. The ubiquitin system. Annu. Rev. Biochem. 67: 425–479. Ingram, G.C., Goodrich, J., Wilkinson, M.D., Simon, R., Haughn, G.W. and Coen, E.S. 1995. Parallels between UNUSUAL FLORAL ORGANS and FIMBRIATA, genes controlling flower development in Arabidopsis and Antirrhinum. Plant Cell 7: 1501–1510. Kamura, T., Conrad, M.N., Yan, Q., Conaway, R.C. and Conaway, J.W. 1999. The Rbx1 subunit of SCF and VHL E3 ubiquitin ligase activates Rub1 modification of cullins Cdc53 and Cul2. Genes Dev. 13: 2928–2933. Knight, J. 2002. All genomes great and small. Nature 417: 374–376. Koepp, D.M., Harper, J.W. and Elledge, S.J. 1999. How the cyclin became a cyclin: regulated proteolysis in the cell cycle. Cell 97: 431–434. Levin, J.Z., and Meyerowitz, E.M. 1995. UFO: an Arabidopsis gene involved in both floral meristem and floral organ development. Plant Cell 7: 529–548. Moffatt, B.A., McWhinnie, E.A., Agarwal, S.K. and Schaff, D.A. 1994. The adenine phosphoribosyltransferase-encoding gene of Arabidopsis thaliana. Gene 143: 211–216. Porat, R., Lu, P. and O’Neill, S.D. 1998. Arabidopsis SKP1, a homologue of a cell cycle regulator gene, is predominantly expressed in meristematic cells. Planta 204: 345–351. Risseeuw, E.P., Daskalchuk, T.E., Banks, T.W., Liu, E., Cotelesage, J., Hellmann, H., Estelle, M., Somers, D.E. and Crosby, W.L. 2003. Protein interaction analysis of SCF ubiquitin E3 ligase subunits from Arabidopsis. Plant J. 34: 753–767. Ross, K.J., Fransz, P. and Jones, G.H. 1996. A light microscopic atlas of meiosis in Arabidopsis thaliana. Chromosome Res. 4: 507–516. Ruegger, M., Dewey, E., Gray, W.M., Hobbie, L., Turner, J. and Estelle, M. 1998. The TIR1 protein of Arabidopsis functions in auxin response and is related to human SKP2 and yeast grr1p. Genes Dev. 12: 198–207. Samach, A., Klenz, J.E., Kohalmi, S.E., Risseeuw, E., Haughn, G.W. and Crosby, W.L. 1999. The UNUSUAL FLORAL ORGANS gene of Arabidopsis thaliana is an F-box protein required
173 for normal patterning and growth in the floral meristem. Plant J. 20: 433–445. Schulman, B.A., Carrano, A.C., Jeffrey, P.D., Bowen, Z., Kinnucan, E.R., Finnin, M.S., Elledge, S.J., Harper, J.W., Pagano, M. and Pavletich, N.P. 2000. Insights into SCF ubiquitin ligases from the structure of the Skp1-Skp2 complex. Nature 408: 381–386. Schwechheimer, C., Serino, G., Callis, J., Crosby, W.L., Lyapina, S., Deshaies, R.J., Gray, W.M., Estelle, M. and Deng, X.W. 2001. Interactions of the COP9 signalosome with the E3 ubiquitin ligase SCFTIRI in mediating auxin response. Science 292: 1379–1382. Seol, J.H., Feldman, R.M., Zachariae, W., Shevchenko, A., Correll, C.C., Lyapina, S., Chi, Y., Galova, M., Claypool, J., Sandmeyer, S., Nasmyth, K. and Deshaies, R.J. 1999. Cdc53/cullin and the essential Hrt1 RING-H2 subunit of SCF define a ubiquitin ligase module that activates the E2 enzyme Cdc34. Genes Dev. 13: 1614–1626. Skowyra, D., Craig, K.L., Tyers, M., Elledge, S.J. and Harper, J.W. 1997. F-box proteins are receptors that recruit phosphorylated substrates to the SCF ubiquitin-ligase complex. Cell 91: 209– 219. Xie, D.X., Feys, B.F., James, S., Nieto-Rostro, M. and Turner, J.G. 1998. COI1: an Arabidopsis gene required for jasmonateregulated defense and fertility. Science 280: 1091–1094.
Xu, L., Liu, F., Lechner, E., Genschik, P., Crosby, W.L., Ma, H., Peng, W., Huang, D. and Xie, D. 2002. The SCFCOI1 ubiquitin-ligase complexes are required for jasmonate response in Arabidopsis. Plant Cell 14: 1919–1935. Yang, M., Hu, Y., Lodhi, M., McCombie, W.R. and Ma, H. 1999. The Arabidopsis SKP1-LIKE1 gene is essential for male meiosis and may control homologue separation. Proc. Natl. Acad. Sci. USA 96: 11416–11421. Zhao, D., Yang, M., Solava, J. and Ma, H. 1999. The ASK1 gene regulates development and interacts with the UFO gene to control floral organ identity in Arabidopsis. Dev. Genet. 25: 209–223. Zhao, D., Yu, Q., Chen, M. and Ma, H. 2001. The ASK1 gene regulates B function gene expression in cooperation with UFO and LEAFY in Arabidopsis. Development 128: 2735–2746. Zhao, D., Ni, W., Feng, B., Han, T., Petrasek, M.G. and Ma, H. 2003. Members of the ASK gene family exhibit a variety of expression patterns and may play diverse roles in Arabidopsis. Plant Physiol. 133: 203–217. Zheng, N., Schulman, B.A., Song, L., Miller, J.J., Jeffrey, P.D., Wang, P., Chu, C., Koepp, D.M., Elledge, S.J., Pagano, M., Conaway, R.C., Conaway, J.W., Harper, J.W. and Pavletich, N.P. 2002. Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase complex. Nature 416: 703–709.
